Molecular Taxonomic Evidence for Two Distinct Genotypes of Mycobacterium yongonense via Genome-Based Phylogenetic Analysis

Recently, we introduced a distinct Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950^T and Mycobacterium yongonense DSM 45126^T. Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126^T than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes (argH, dnaA, deaD, hsp65, and recF) and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum.     

Comparative Genome Analysis of Mycobacterium avium Revealed Genetic Diversity in Strains that Cause Pulmonary and Disseminated Disease

Mycobacterium avium complex (MAC) infection causes disseminated disease in immunocompromised hosts, such as human immunodeficiency virus (HIV)-positive patients, and pulmonary disease in persons without systemic immunosuppression, which has been increasing in many countries. In Japan, the incidence of pulmonary MAC disease caused by M. avium is about 7 times higher than that caused by M. intracellulare. To explore the bacterial factors that affect the pathological state of MAC disease caused by M. avium, we determined the complete genome sequence of the previously unreported M. avium subsp. hominissuis strain TH135 isolated from a HIV-negative patient with pulmonary MAC disease and compared it with the known genomic sequence of M. avium strain 104 derived from an acquired immunodeficiency syndrome patient with MAC disease. The genome of strain TH135 consists of a 4,951,217-bp circular chromosome with 4,636 coding sequences. Comparative analysis revealed that 4,012 genes are shared between the two strains, and strains TH135 and 104 have 624 and 1,108 unique genes, respectively. Many strain-specific regions including virulence-associated genes were found in genomes of both strains, and except for some regions, the G+C content in the specific regions was low compared with the mean G+C content of the corresponding chromosome. Screening of clinical isolates for genes located in the strain-specific regions revealed that the detection rates of strain TH135-specific genes were relatively high in specimens isolated from pulmonary MAC disease patients, while, those of strain 104-specific genes were relatively high in those from HIV-positive patients. Collectively, M. avium strains that cause pulmonary and disseminated disease possess genetically distinct features, and it suggests that the acquisition of specific genes during strain evolution has played an important role in the pathological manifestations of MAC disease.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Comparison of the Virulence for Mice of Mycobacterium avium and Mycobacterium intracellulare Identified by DNA Probe Test

The virulence of various serovars of Mycobacterium avium and M. intracellulare identified by DNA probe test was compared with each other. We found species- and serovar-dependencies of M. avium complex (MAC) virulence to mice in terms of mortality, incidence of lung lesions and bacterial load in the visceral organs, as follows. First, human- or environment-derived M. intracellulare was more virulent for mice, as compared to M. avium isolated from patients or environmental sources. Second, the virulence of MAC isolates belonging to serovars 1, 8, 9 (M. avium), 14 and 16 (M. intracellulare) is in the order of serovars 16 > 14 > 8 > 1 > 9. These aspects were different from those for MAC virulence to human and bird, swine and cattle.     

Performance Assessment of the BluePoint MycoID Plus Kit for Identification of Mycobacterium tuberculosis,Including Rifampin- and Isoniazid-resistant Isolates,and Nontuberculous Mycobacteria

The performance of the BluePoint MycoID plus kit (Bio Concept Corporation, Taichung, Taiwan), which was designed to simultaneously detect Mycobacterium tuberculosis (MTB), rifampin- and isoniazid-resistant MTB, and nontuberculous mycobacteria (NTM) was first evaluated with 950 consecutive positive cultures in Mycobacterium Growth Indicator Tube (MGIT) system (BACTEC, MGIT 960 system, Becton-Dickinson, Sparks) from clinical respiratory specimens. The discrepant results between kit and culture-based identification were finally assessed by 16S rRNA gene sequencing and clinical diagnosis. The accuracy rate of this kit for identification of all Mycobacterium species was 96.3% (905/940). For MTB identification, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the kit were 99.7%, 99.3%, 99.0% and 99.8%, respectively. For rifampicin-resistant MTB identification, the sensitivity, specificity, PPV, and NPV of the kit were 100.0%, 99.4%, 91.3%, and 100.0%, respectively, while the corresponding values of isoniazid-resistant MTB identification were 82.6%, 99.4%, 95.0%, and 97.6%, respectively. In identifying specific NTM species, the kit correctly identified 99.3% of M. abscessus (147/148) complex, 100% of M. fortuitum (32/32), M. gordonae (38/38), M. avium (39/39), M. intracellulare (90/90), M. kansasii (36/36), and M. avium complex species other than M. avium and M. intracellulare (94/94). In conclusions, the diagnostic value of the BluePoint MycoID plus kit was superior to culture method for recoveries and identification of NTM to species level. In addition, the diagnostic accuracy of BluePoint MycoID plus kit in MTB identification was similar to conventional culture method with high accuracy rate of rifampicin-resistant M. tuberculosis identification.     

Fluorescent Acid-Fast Microscopy for Measuring Phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and Their Intracellular Growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30°C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.     

Limited Contribution of IL-36 versus IL-1 and TNF Pathways in Host Response to Mycobacterial Infection

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M. bovis BCG infection and virulent aerogenic M. tuberculosis infection. IL-36γ expression was increased in the lung of M. bovis BCG infected mice. However, IL-36R deficient mice infected with M. bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M. tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection.     

TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.     

Mycobacterium chimaera as an Underestimated Cause of NTM Lung Diseases in Patients Hospitalized in Pulmonary Wards

Mycobacterium chimaera is the newly described species belonging to Mycobacterium avium complex (MAC), with morphology and growth characteristics closely related to Mycobacterium intracellulare. The aim of this retrospective study was to analyze the frequency and clinical significance of M. chimaera identification in the population of patients with previous positive respiratory cultures for M. intracellulare or MAC. 200 strains of M. intracellulare or MAC, isolated from respiratory specimens of patients hospitalized in pulmonary wards, between 2011 and 2020, were retrospectively analyzed with GenoType NTM-DR test. 88 (44%) of strains were re-classified to M. chimaera species. Analysis of clinical data in 30 patients with positive M. chimaera isolates revealed that they were diagnosed with chronic obstructive pulmonary disease (COPD) – 27%, past tuberculosis – 20%, or interstitial lung diseases – 17%, respectively. Non-tuberculous mycobacterial lung disease (NTMLD) caused by M. chimaera has been recognized in 53% of patients, most often in those presenting with post-tuberculous lung lesions. M. chimaera was almost exclusively isolated from respiratory specimens of patients with underlying lung diseases, especially those with COPD and/or past tuberculosis. NTMLD due to M. chimaera was diagnosed predominantly in patients with past tuberculosis.     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.     

Association of ISMav6 with the Pattern of Antibiotic Resistance in Korean Mycobacterium avium Clinical Isolates but No Relevance between Their Genotypes and Clinical Features

The aim of this study was to genetically characterize clinical isolates from patients diagnosed with Mycobacterium avium lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of M. avium isolates from 92 Korean patients revealed that all isolates were M. avium subspecies hominissuis. In hsp65 sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS1311 positive and IS900 negative. Four of the M. avium subsp. hominissuis isolates did not harbor IS1245 and 1 of the M. avium isolates intriguingly harbored DT1, which is thought to be a M. intracellulare-specific element. M. avium subsp. hominissuis harboring ISMav6 is prevalent in Korea. No significant association between clinical manifestation and treatment response has been found in patients with the hsp65 code type and ISMav6, indicating that no specific strain/genotype among M. avium subsp. hominissuis organisms was a major source of M. avium lung disease. Interestingly, the presence of ISMav6 was correlated with greater resistance to moxifloxacin. Conclusively, the genotype of Korean M. avium subsp. hominissuis isolates is not a disease determinant responsible for lung disease and specific virulent factors of M. avium subsp. hominissuis need to be investigated further.     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Latent Microsporidiosis Caused by Encephalitozoon cuniculi in Immunocompetent Hosts: A Murine Model Demonstrating the Ineffectiveness of the Immune System and Treatment with Albendazole

Background

Microsporidia are obligate intracellular parasites causing severe infections with lethal outcome in immunocompromised hosts. However, these pathogens are more frequently reported as latent infections in immunocompetent individuals and raises questions about the potential risk of reactivation following induced immunosuppression.

Aims

To evaluate the possibility latent microsporidiosis, efficacy or albendazole, and reactivation, the authors monitored the course of E. cuniculi infection in immunocompetent BALB/c mice and immunodeficient SCID mice using molecular methods.

Methods

Mice were per orally infected with 10⁷ spores of E. cuniculi. Selected groups were treated with albendazole, re-infected or chemically immunosuppressed by dexamethasone. The presence of microsporidia in the host’s organs and feces were determined using PCR methods. Changes in numbers of lymphocytes in blood and in spleen after induction of immunosuppression were confirmed using flow cytometry analysis.

Results

Whereas E. cuniculi caused lethal microsporidiosis in SCID mice, the infection in BABL/c mice remained asymptomatic despite parasite dissemination into many organs during the acute infection phase. Albendazole treatment led to microsporidia elimination from organs in BALB/c mice. In SCID mice, however, only a temporary reduction in number of affected organs was observed and infection re-established post-treatment. Dexamethasone treatment resulted in a chronic microsporidia infection disseminating into most organs in BALB/c mice. Although the presence of E. cuniculi in organs of albendazole- treated mice was undetectable by PCR, it was striking that infection was reactivated by immunosuppression treatment.

Conclusion

Our results demonstrated that microsporidia can successfully survive in organs of immunocompetent hosts and are able to reactivate from undetectable levels and spread within these hosts after induction of immunosuppression. These findings stress the danger of latent microsporidiosis as a life-threatening risk factor especially for individuals undergoing chemotherapy and in transplant recipients of organs originating from infected donors.     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Decorin Binding Proteins of Borrelia burgdorferi Promote Arthritis Development and Joint Specific Post-Treatment DNA Persistence in Mice

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.     

Reactivation of Latent Tuberculosis in Cynomolgus Macaques Infected with SIV Is Associated with Early Peripheral T Cell Depletion and Not Virus Load

HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.     

Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis ‘knock-in’ strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.     

Ion Channel Blockers as Antimicrobial Agents,Efflux Inhibitors,and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.     

Identification of antibody responses to the serotype-nonspecific molecular species of glycopeptidolipids in Mycobacterium avium infection

Glycopeptidolipids (GPLs) comprise a major surface glycolipid of Mycobacterium avium complex (MAC), and their unique oligosaccharide extensions are known to define MAC serotypes. Beside the mature form of “serotype-specific” GPLs (ssGPLs), those that share the backbone structure but lack the oligosaccharide extensions exist as abundantly in all MAC serotypes, but the presumption was that antibody responses might not be directed to these “serotype-nonspecific” GPLs (nsGPLs) due to the lack of the sugar chain epitope. Here, we show that IgG responses to nsGPLs indeed occur in MAC-infected guinea pigs. The pool of anti-nsGPL antibodies was distinct from that of anti-ssGPL antibodies in terms of requirements for the oligosaccharide and acetylation for their target recognition. Because nsGPLs are shared in virtually all MAC strains, but totally absent in Mycobacterium tuberculosis, this study suggests that detecting serum anti-nsGPL antibodies can potentially be useful for differential diagnosis of MAC infection and tuberculosis.     

Factors Influencing Numbers of Mycobacterium avium, Mycobacterium intracellulare, and Other Mycobacteria in Drinking Water Distribution Systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter⁻¹. The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r² = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm²). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.