Pathological granuloma fibrosis induced by agar-embedded Mycobacterium abscessus in C57BL/6JNarl mice.

Introduction: Pulmonary granuloma diseases caused by Mycobacterium abscessus (M. abscessus) have increased in past decades, and drug-resistance in this pathogen is a growing public health concern. Therefore, an animal model of chronic granuloma disease is urgently needed. Methods: In this study, M. abscessus embedded within agar beads (agar-AB) was used to develop such a model in C57BL/6JNarl mice. Results: Chronic infection was sustained for at least 3 months after agar-AB infection, visible granulomas spread in the lungs, and giant cells and foamy cells appeared in the granulomas. More importantly, pulmonary fibrosis progressed for 3 months, and collagen fibers were detected by Masson trichrome staining. Further, inducible nitric oxide synthase (iNOS) was highly expressed within the alveolar space, and the fibrosis-mediator transforming growth factor beta (TGF-ß) began to be expressed at 1 month. Hypoxia-inducible factor (HIF-1α) expression also increased, which aided in normalizing oxygen partial pressure. Discussion: Although the transient fibrosis persisted for only 3 months, and the pulmonary structure resolved when the pathogen was cleard, this pulmonary fibrosis model for M. abscessus infection will provide a novel test platform for development of new drugs, regimens, and therapies.

Whole Genome Sequencing and Pan-Genomic Analysis of Multidrug-Resistant Vibrio cholerae VC01 Isolated from a Clinical Sample.

Cholera, a disease caused by the Vibrio cholerae bacteria, threatens public health worldwide. The organism mentioned above has a significant historical record of being identified as a prominent aquatic environmental pollutant capable of adapting its phenotypic and genotypic traits to react to host patients effectively. This study aims to elucidate the heterogeneity of the sporadic clinical strain of V. cholerae VC01 among patients residing in Silvasa. The study involved conducting whole-genome sequencing of the isolate obtained from patients exhibiting symptoms, including those not commonly observed in clinical practice. The strain was initially identified through a combination of biochemical analysis, microscopy, and 16s rRNA-based identification, followed by type strain-based identification. The investigation demonstrated the existence of various genetic alterations and resistance profiles against multiple drugs, particularly chloramphenicol (catB9), florfenicol (floR), oxytetracycline (tet(34)), sulfonamide (sul2), and Trimethoprim (dfrA1). The pan-genomic analysis indicated that 1099 distinct clusters were detected within the genome sequences of recent isolates worldwide. The present study helps to establish a correlation between the mutation and the coexistence of antimicrobial resistance toward current treatment.

Microbiota dysbiosis in odontogenic rhinosinusitis and its association with anaerobic bacteria.

Odontogenic rhinosinusitis is a subtype of rhinosinusitis associated with dental infection or dental procedures and has special bacteriologic features. Previous research on the bacteriologic features of odontogenic rhinosinusitis has mainly used culture-dependent methods. The variation of microbiota between odontogenic and nonodontogenic rhinosinusitis as well as the interplay between the involved bacteria have not been explored. Therefore, we enrolled eight odontogenic rhinosinusitis cases and twenty nonodontogenic rhinosinusitis cases to analyze bacterial microbiota through 16S rRNA sequencing. Significant differences were revealed by the Shannon diversity index (Wilcoxon test p = 0.0003) and PERMANOVA test based on weighted UniFrac distance (Wilcoxon test p = 0.001) between odontogenic and nonodontogenic samples. Anaerobic bacteria such as Porphyromonas, Fusobacterium, and Prevotella were significantly dominant in the odontogenic rhinosinusitis group. Remarkably, a correlation between different bacteria was also revealed by Pearson's correlation. Staphylococcus was highly positively associated with Corynebacterium, whereas Fusobacterium was highly negatively correlated with Prophyromonas. According to our results, the microbiota in odontogenic rhinosinusitis, predominantly anaerobic bacteria, was significantly different from that in nonodontogenic rhinosinusitis, and the interplay between specific bacteria may a major cause of this subtype of rhinosinusitis.

Suppression of SREBP-1 Expression by Simvastatin Decreases Visfatin-Induced Chemoresistance to Sunitinib in Human Renal Carcinoma 786-O Cells.

The resistance of renal cell carcinoma (RCC) to sunitinib impedes the success of chemotherapy in cancer treatment. Although several sunitinib resistance mechanisms have been proposed, little is known concerning the impact of obesity and adipokines in RCC cells. The upregulation of sterol-regulatory element-binding protein-1 (SREBP-1) has been reported to modulate the progression of tumor cells. The present study investigated the effect of visfatin on sunitinib-induced cytotoxicity in RCC cells through SREBP-1 expression. We found that visfatin-induced Akt and p70S6K activation increased SREBP-1 expression in 786-O cells. The visfatin-induced SREBP-1 mRNA and protein levels were attenuated through the inactivation of Akt and p70S6K by pharmacological inhibitors. In addition, the SREBP-1 knockdown using siRNA enhanced the cytotoxic effects of sunitinib. Our results also revealed the roles of simvastatin in attenuating the effects of visfatin on 786-O cells by inhibiting the production of reactive oxygen species. In particular, simvastatin co-treatment increased the cell cytotoxicity of sunitinib in visfatin-treated 786-O cells, which were associated with down-regulation of SREBP-1 expression. Our results suggest an important role of SREBP-1 in visfatin-induced drug resistance of RCC cells to sunitinib. The cytotoxic mechanism of simvastatin on RCC cells may provide a new strategy to improve therapeutic outcomes for the RCC treatment.

Fermented Ginger Extract in Natural Deep Eutectic Solvent Enhances Cytotoxicity by Inhibiting NF-κB Mediated CXC Chemokine Receptor 4 Expression in Oxaliplatin-Resistant Human Colorectal Cancer Cells.

Ginger extracts have been shown to have health-promoting pharmacological activity and beneficial effects, including antioxidant and anticancer properties. The extraction of ginger by natural deep eutectic solvents (NaDES) has been shown to enhance bioactivity, but the cytotoxicity of NaDES extracts needs to be further determined. Signaling through the CXC chemokine receptor 4 (CXCR4) expressed on colorectal cancer (CRC) cells has a pivotal role in tumor cell chemosensitivity. Oxaliplatin is a third-generation platinum compound used as an effective chemotherapeutic drug for CRC treatment. However, whether ginger extract and oxaliplatin could induce a synergistic cytotoxic effect in oxaliplatin-resistant CRC cells through modulating CXCR4 expression is not known. In this study, oxaliplatin-resistant HCT-116 (HCT-116/R) cells were generated first. Ginger was extracted using the NaDES mixture betaine/lactate/water (1:2:2.5). Lactobacillus reuteri fermentation of NaDES-ginger extract increased the total polyphenol content (12.42 mg gallic acid/g in non-fermented NaDES-ginger extract and 23.66 mg gallic acid/g in fermented NaDES-ginger extract). It also increased the antioxidant activity by about 20−30% compared to non-fermented NaDES-ginger extract. In addition, it achieved low cytotoxicity to normal colonic mucosal cells and enhanced the anticancer effect on HCT-116/R cells. On the other hand, the inhibition of NF-κB activation by fermented NaDES-ginger extract significantly decreased the CXCR4 expression (p < 0.05) in HCT-116/R cells. The inactivation of NF-κB by pharmacological inhibitor pyrrolidine dithiocarbamate further enhanced the fermented NaDES-ginger extract-reduced CXCR4 expression levels (p < 0.05). Moreover, fermented NaDES-ginger extract could synergistically increase the cytotoxicity of oxaliplatin by inhibiting CXCR4 expression and inactivating NF-κB, resulting in HCT-116/R cell death. These findings demonstrate that fermented NaDES-ginger extract reduces the NF-kB-mediated activation of CXCR4 and enhances oxaliplatin-induced cytotoxicity in oxaliplatin-resistant CRC cells.

Biologically Synthesized Silver Nanoparticles and Their Diverse Applications.

Nanotechnology has become the most effective and rapidly developing field in the area of material science, and silver nanoparticles (AgNPs) are of leading interest because of their smaller size, larger surface area, and multiple applications. The use of plant sources as reducing agents in the fabrication of silver nanoparticles is most attractive due to the cheaper and less time-consuming process for synthesis. Furthermore, the tremendous attention of AgNPs in scientific fields is due to their multiple biomedical applications such as antibacterial, anticancer, and anti-inflammatory activities, and they could be used for clean environment applications. In this review, we briefly describe the types of nanoparticle syntheses and various applications of AgNPs, including antibacterial, anticancer, and larvicidal applications and photocatalytic dye degradation. It will be helpful to the extent of a better understanding of the studies of biological synthesis of AgNPs and their multiple uses.

Alginate-Capped Silver Nanoparticles as a Potent Anti-mycobacterial Agent Against Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb). Although progress has been made in TB control, still about 10 million people worldwide develop TB annually and 1.5 million die of the disease. The rapid emergence of aggressive, drug-resistant strains and latent infections have caused TB to remain a global health challenge. TB treatments are lengthy and their side effects lead to poor patient compliance, which in turn has contributed to the drug resistance and exacerbated the TB epidemic. The relatively low output of newly approved antibiotics has spurred research interest toward alternative antibacterial molecules such as silver nanoparticles (AgNPs). In the present study, we use the natural biopolymer alginate to serve as a stabilizer and/or reductant to green synthesize AgNPs, which improves their biocompatibility and avoids the use of toxic chemicals. The average size of the alginate-capped AgNPs (ALG-AgNPs) was characterized as nanoscale, and the particles were round in shape. Drug susceptibility tests showed that these ALG-AgNPs are effective against both drug-resistant Mtb strains and dormant Mtb. A bacterial cell-wall permeability assay showed that the anti-mycobacterial action of ALG-AgNPs is mediated through an increase in cell-wall permeability. Notably, the anti-mycobacterial potential of ALG-AgNPs was effective in both zebrafish and mouse TB animal models in vivo. These results suggest that ALG-AgNPs could provide a new therapeutic option to overcome the difficulties of current TB treatments.

Characterization of Virus Replication, Pathogenesis, and Cytokine Responses in Syrian Hamsters Inoculated with SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus which caused a global respiratory disease pandemic beginning in December 2019. Understanding the pathogenesis of infection and the immune responses in a SARS-CoV-2-infected animal model is urgently needed for vaccine development. METHODS: Syrian hamsters (Mesocricetus auratus) were intranasally inoculated with 105, 5×105, and 106 TCID50 of SARS-CoV-2 per animal and studied for up to 14 days. Body weight, viral load and real-time PCR amplification of the SARS-CoV-2 N gene were measured. On days 3, 6 and 9, lung, blood, liver, pancreas, heart, kidney, and bone marrow were harvested and processed for pathology, viral load, and cytokine expression. RESULTS: Body weight loss, increased viral load, immune cell infiltration, upregulated cytokine expression, viral RNA, SARS-CoV-2 nucleoprotein, and mucus were detected in the lungs, particularly on day 3 post-infection. Extremely high expression of the pro-inflammatory cytokines MIP-1 and RANTES was detected in lung tissue, as was high expression of IL-1ß, IL-6, IL-12, and PD-L1. The glutamic oxalacetic transaminase/glutamic pyruvic transaminase (GOT/GPT) ratio in blood was significantly increased at 6 days post-infection, and plasma amylase and lipase levels were also elevated in infected hamsters. CONCLUSION: Our results provide new information on immunological cytokines and biological parameters related to the pathogenesis and immune response profile in the Syrian hamster model of SARS-CoV-2 infection.

Corrigendum: Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

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Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette-Guérin (BCG) vaccine against Mycobacterium tuberculosis (MTB) has been available for decades, its efficacy is variable and has lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.

Dynamics of the lung microbiome in intensive care patients with chronic obstructive pulmonary disease and community-acquired pneumonia.

Little is known about the composition and clinical implications of lung microbiome in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia requiring invasive mechanical ventilation and intensive care unit admission. Therefore, this study aimed to explore the longitudinal changes in microbial airway composition and its variations between COPD patients with different weaning outcomes. Fifty-one endotracheal aspirate samples from 21 participants and 5 saline samples were collected as the patient and control group, respectively. Sequence analysis revealed significant increases and upward trends in the relative abundance of the Acinetobacter genus and Acinetobacter baumannii complex species in paired comparisons of sampling points and over time, respectively, in patients with failed weaning (p for trend = 0.012 and 0.012, respectively) but not in those with successful weaning (p for trend = 0.335 and 0.426, respectively). Furthermore, significant changes in the composition of the bacterial community were observed in paired comparisons of sampling points in patients with failed weaning compared with those with successful weaning. The alpha diversity did not differ between the patients with different weaning outcomes. These results further the understanding of longitudinal airway microbiome structure analysis and its clinical implications when managing critically ill patients with and without COPD.

Urolithiasis Is a Risk Factor for Uroseptic Shock and Acute Kidney Injury in Patients With Urinary Tract Infection.

Urinary tract infection (UTI) is a common complication in patients with urolithiasis. This study aimed to compare clinical manifestations and treatment outcomes among UTI patients with or without urolithiasis. It also focused on identifying relationships among urolithiasis, uroseptic shock, and acute kidney injury (AKI). This retrospective study enrolled hospitalized UTI patients who underwent imaging in an acute care setting from January 2006 to March 2015. Of 662 participants enrolled, 113 (17.1%) had urolithiasis, 107 (16.2%) developed uroseptic shock, and 184 (27.8%) developed AKI. A multivariate logistic regression analysis showed that in UTI patients, urolithiasis is associated with an increased risk of uroseptic shock (OR 1.80, 95% CI: 1.08-3.02, P = 0.025), AKI (OR 1.95, 95% CI: 1.22-3.12, P = 0.005), and bacteremia (OR 1.68, 95% CI: 1.08-2.64, P = 0.022). Urolithiasis is common in UTI patients and is associated with an increased risk of uroseptic shock and AKI.

Microbiota Dysbiosis in Fungal Rhinosinusitis.

Fungal rhinosinusitis is a unique phenotype of chronic rhinosinusitis with unique clinical and histological characteristics. The role of bacterial microbiota in various phenotypes chronic rhinosinusitis is not thoroughly understood. Therefore, we conducted 16s rRNA amplification sequencing to determine differences in bacterial communities between phenotypes (fungal vs. non- fungal) and anatomical sites (middle meatus vs. nasopharynx). Endoscope-guided swabs were used to collect samples from the middle meatus and nasopharynx of seven consecutive patients with fungal and 18 consecutive patients with non-fungal rhinosinusitis. DNA was extracted and investigated through 16S rRNA amplification. Among samples from the middle meatus, Shannon diversity was significantly lower in those from the fungal rhinosinusitis group (p = 0.029). However, no significant differences in diversity were noted between nasopharynx samples (p = 0.85). Fungal rhinosinusitis samples exhibited a distinct distribution of taxon relative abundance, which involved not only the absence of rhinosinusitis-associated commensal Corynebacterium and Fusobacterium in the middle meatus but also a significant increase in Haemophilus prevalence and abundance. This is the first study to compare bacterial communities in fungal and non-fungal rhinosinusitis samples. Our findings demonstrated that bacterial community dysbiosis was more apparent in fungal rhinosinusitis samples and was limited to the middle meatus.

Rapid identification of capsular serotype K1/K2 Klebsiella pneumoniae in pus samples from liver abscess patients and positive blood culture samples from bacteremia cases via an immunochromatographic strip assay.

BACKGROUND: In Asia, serotype K1/K2 Klebsiella pneumoniae are the major capsular serotypes that cause liver abscess or bacteremia in patients. The purpose of this study was to compare novel immunochromatographic strips (ICSs), which can rapidly detect K. pneumoniae serotypes K1/K2 in clinical samples, to conventional capsular serotyping methods. METHODS: Pus drainage samples from 16 patients with a liver abscess caused by K. pneumoniae, blood samples from 112 positive flagged blood culture bottle and a subsequent single colony in the medium were tested with the ICS. The results were then compared to findings of capsular swelling tests. Samples subjected to the polymerase chain reaction (PCR) analysis were used as reference. RESULTS: The identification of K. pneumoniae via the traditional bacterial culture from pus samples took 3.4 days on average (ranging from 2.2 to 5.5 days). Further capsular serotyping of K. pneumoniae by the capsular swelling test of pure isolates lasted 5-10 min, and the PCR method took ~ 4 h. As for ICSs, the time for direct identification of the K. pneumoniae capsular serotype K1/K2 in pus was < 4 min (ranging from 2 to 4 min). The results of ICSs were consistent with capsular swelling tests and PCR methods. Testing of 112 blood culture samples and subsequent single colonies in the medium with ICSs yielded consistent results for most samples. CONCLUSIONS: This study indicates that ICSs can rapidly detect K. pneumoniae serotypes K1 and K2 in pus or positive flagged blood culture broth samples within 5 min. Their accuracy is comparable to that of the conventional capsular serotyping methods such as a serum agglutination assay or PCR.

Production of N-acetyl-D-neuraminic Acid by Recombinant Single Whole Cells Co-expressing N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic Acid Aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) is a costly precursor for many drugs such as anti-influenza antivirals. In a previous study, a whole-cell process for Neu5Ac production was developed using a combination of two Escherichia coli cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine-2-epimerase (bage) and E. coli N-acetyl-D-neuraminic acid aldolase (nanA), respectively. In this study, we constructed a bAGE and NanA co-expression system to improve Neu5Ac production. Two recombinant E. coli strains, E. coli BL21 (DE3) pET-bage-nanA (HA) and E. coli BL21 (DE3) pET-bage-2nanA (HAA), synchronously expressing bAGE and NanA were used as biocatalysts to generate Neu5Ac from N-acetyl-D-glucosamine (GlcNAc) and pyruvate. The HA biocatalysts produced 187.5 mM Neu5Ac within 8 h. The yield of GlcNAc was 15.6%, and the Neu5Ac production rate was 7.25 g/L/h. The most active HAA biocatalysts generated 412.6 mM Neu5Ac and a GlcNAc yield of 34.4%. HAA achieved a Neu5Ac production rate of 15.9 g/L/h, which surpassed those for all reported Neu5Ac production processes so far. The present study demonstrates that using recombinant E. coli cells synchronously expressing bAGE and NanA as biocatalysts could potentially be used in the industrial mass production of Neu5Ac.

Phosphoenolpyruvate phosphotransferase system components positively regulate Klebsiella biofilm formation.

BACKGROUND/PURPOSE: Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs), which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes) consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates diverse processes of bacterial physiology. In the genome of K. pneumoniae MGH 78578, we found an uncharacterized enzyme II complex homolog of PTS: KPN00353 (EIIA homolog), KPN00352 (EIIB homolog), and KPN00351 (EIIC homolog). The aim of this study was to characterize the potential physiological role of KPN00353, KPN00352, and KPN00351 in biofilm formation by K. pneumoniae. METHODS/RESULTS: We constructed the PTS mutants and recombinant strains carrying the gene(s) of PTS. The recombinant K. pneumoniae strain overexpressing KPN00353-KPN00352-KPN00351 produced more extracellular matrix than did the vector control according to transmission and scanning electron microscopy. Judging by quantification of biofilm formation, of extracellular DNA (eDNA), and of capsular polysaccharide, the recombinant strain overexpressing KPN00353-KPN00352-KPN00351 produced more biofilm and capsular polysaccharide after overnight culture and more eDNA in the log phase as compared to the vector control. CONCLUSION: The genes, KPN00353-KPN00352-KPN00351, encode a putative enzyme II complex in PTS and positively regulate biofilm formation by enhancing production of eDNA and capsular polysaccharide in K. pneumoniae. Five proteins related to chaperones, to the citric acid cycle, and to quorum sensing are upregulated by the KPN00353-KPN00352-KPN00351 system.

Combining molecular typing and spatial pattern analysis to identify areas of high tuberculosis transmission in a moderate-incidence county in Taiwan.

In total, 303 randomly selected clinical Mycobacterium tuberculosis (MTB) isolates from 303 patients (collected January to December 2012) in central Taiwan were examined. The major lineages found were Beijing (N = 114, 37.62%), Haarlem (N = 76, 25.08%) and East African-Indian (EAI) (N = 42, 13.86%). Notably, younger persons (≤30 years old) were 6.58 times more likely to be infected with a Beijing genotype compared to older persons (>70 years) (p < 0.05). Combining molecular typing methods and geographical information system (GIS) analysis, we uncovered a twofold higher incidence of Beijing strains in a hotspot area (33%) compared to non-hotspot areas (17%). By 24 MIRU-VNTR typing, persons in clustered groups were 1.96 times more likely to be infected with a Beijing strain compared with non-clustered persons, suggesting recent spread and emergence of MTB. Finally, we observed a trend in which TB incidence increased as the density/concentration of analyzed environmental factors increased, suggesting that environmental factors are associated with TB transmission; however, only population density was found to be significantly associated with increased risk of TB (p < 0.05). Molecular typing methods combined with spatial analysis suggest possible TB transmission. Early intervention to interrupt transmission may be most effective if targeted to hot zones of TB.

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome sequencing and comparing the results to the sequence of the H37Rv reference strain. One hundred and ten SNPs, each unique to one of the six strains, were used to genotype 150 additional isolates by applying DNA mass spectrometry. Lineage-specific SNPs were identified that could distinguish the major lineages of the clinical isolates. A subset including 32 SNPs was found to be sufficient to type four major groups of MTB isolates in Taiwan (ancient Beijing, modern Beijing, East African-Indian, and Latin-American Mediterranean). However, there was high genetic homozygosity within the Euro-American lineage, which included spoligotype-classified Haarlem and T strains. By whole-genome sequencing of 12 representative Euro-American isolates, we identified multiple subtype-specific SNPs which allowed us to distinguish two major branches within the Euro-American lineage.

Genetic diversity of the Mycobacterium tuberculosis East African-Indian family in three tropical Asian countries.

BACKGROUND: The Beijing lineage of Mycobacterium tuberculosis (MTB) is the most predominant MTB strain in Asian countries and is spreading worldwide, however, the East African-Indian (EAI) lineage is also particularly prevalent in many tropical Asian countries. The evolutionary relationships among MTB EAI isolates from Taiwan and those of tropical Asian countries remain unknown. METHODS: The EAI strains collected from patients in Taiwan were analyzed using spacer oligonucleotide typing and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing, and compared with published profiles from Cambodia and Singapore to investigate potential epidemiological linkages. RESULTS: Among the three countries, the EAI lineage was most prevalent in Cambodia (60%; Singapore, 25.62%; and Taiwan, 21.85%), having also the highest rates of multidrug resistance and lowest rates of clustering of MTB isolates. We describe a convenient method using seven selected MIRU-VNTR loci for first-line typing to discriminate Beijing and EAI lineages. A potential epidemiological linkage in these tropical Asian countries is also discussed based on a minimum-spanning tree constructed using 24 MIRU-VNTR loci of MTB EAI strains. CONCLUSION: This study identified evolutionary relationships among MTB EAI isolates from Taiwan and those of two other tropical Asian countries, Cambodia and Singapore.