Immunogenicity and vaccine potential of clinical isolate Mycobacterium kansasii strain against Mycobacterium tuberculosis infection.

Mycobacterium kansasii is a bacterium included in non-tuberculous mycobacteria (NTM) that can cause lung disease. It shares a significant number of antigens with Mycobacterium tuberculosis (Mtb), suggesting that it has the potential to be used as a tuberculosis (TB) vaccine. Therefore, we subcutaneously vaccinated mice with reference strain, M. kansasii-ATCC12478 [M. kansasii-American Type Culture Collection (ATCC)], and clinically isolated strain, M. kansasii-SM-1 to evaluate potential as a TB vaccine by comparing with bacille Calmette-Guerin (BCG) vaccine. Ten weeks after vaccination, we evaluated immunogenicity of M. kansasii-ATCC and M. kansasii-SM-1, and M. kansasii-SM-1 immunization induces potent Mtb antigen-specific IFN-γ-producing CD4+ T cells than M. kansasii-ATCC. Upon Mtb infection, M. kansasii-SM-1 provided better protection than M. kansasii-ATCC, which was comparable to the efficacy of BCG. These results showed that the clinical strain M. kansasii-SM-1, which exhibits an enhanced Mtb antigen-specific Th1 response, shows greater vaccine efficacy compared to M. kansasii-ATCC. In this study, we demonstrated that vaccine efficacy can vary depending on the strain of M. kansasii and that its efficacy can be comparable to BCG. This suggests that M. kansasii has the potential to be a live TB vaccine candidate.IMPORTANCEMycobacterium kansasii, a non-tuberculous mycobacteria (NTM) species causing lung disease, shares key antigens with Mycobacterium tuberculosis (Mtb), indicating its potential for TB vaccine development. Subcutaneous vaccination of mice with M. kansasii strains reference strain M. kansasii-ATCC12478 [(M. kansasii-American Type Culture Collection (ATCC)] and clinically isolated strain M. kansasii-SM-1 revealed differences in immunogenicity. M. kansasii-SM-1 induced a robust Mtb antigen-specific IFN-γ-producing CD4+ T cell response compared to M. kansasii-ATCC. Additionally, M. kansasii-SM-1 conferred better protection against Mtb infection than M. kansasii-ATCC, which is comparable to bacille Calmette-Guerin (BCG). These findings underscore the variable vaccine efficacy among M. kansasii strains, with M. kansasii-SM-1 exhibiting promising potential as a live TB vaccine candidate, suggesting its comparative effectiveness to BCG.

Biofilm prevention concentration of clarithromycin against clinically relevant species of nontuberculous mycobacteria.

OBJECTIVE: Mycobacterium avium complex (MAC) and Mycobacterium abscessus are a group of nontuberculous mycobacteria (NTM) that have been described as human pathogens. Their ability to develop biofilms in tissues and medical devices is one of the most important pathogenicity factors, with important implications in diagnosis and treatment. Macrolides are usually considered one of the bases of this treatment. METHODS: Here we have studied the biofilm prevention concentration (BPC) of 16 strains (n=16) with clarithromycin to avoid the biofilm development by these NTM. RESULTS: In this study, all M. abscessus strains have similar BPC, while MAC strains showed different values. For MAC the concentrations ranged between 1-16 mg/L, while for M. abscessus the concentration was 32 mg/L for all strains except one that was 64 mg/L. CONCLUSIONS: These results open the possibility of using macrolides for the prevention of biofilm development in patients with a risk of developing NTM disease.

Mitigation of nontuberculous mycobacteria in hospital water: challenges for infection prevention.

PURPOSE OF REVIEW: The purpose of this review is to summarize recent literature on nontuberculous mycobacteria in water of healthcare systems. Despite improvement in identification techniques and emergence of infection prevention and control programs, nontuberculous mycobacteria remain present in hospital water systems, causing outbreaks and pseudo-outbreaks in healthcare settings. RECENT FINDINGS: Waterborne outbreaks and pseudo-outbreaks of nontuberculous mycobacteria continue to affect hospitals. Improvements in methods of identification and investigation, including MALDI-TOF and whole genome sequencing with evaluation of single nucleotide polymorphisms, have been used successfully in outbreak and pseudo-outbreak investigations. Recent studies have shown control of outbreaks in immunocompromised patients through the use of sterile water for consumption, as well as control of pseudo-outbreaks by using sterile water for procedures. Construction activities have been implicated in outbreaks and pseudo-outbreaks of nontuberculous mycobacteria. Water management programs are now required by the Joint Commission, which will likely improve water risk mitigation. SUMMARY: Improvement in detection and identification of nontuberculous mycobacteria has led to increasing recognition of waterborne outbreaks and pseudo-outbreaks. Water management programs are of vital importance in infection prevention.

Mycobacterium chimaera infections among cardiothoracic surgery patients associated with heater-cooler devices-Kansas and California, 2019.

BACKGROUND: In 2015, an international outbreak of Mycobacterium chimaera infections among patients undergoing cardiothoracic surgeries was associated with exposure to contaminated LivaNova 3T heater-cooler devices (HCDs). From June 2017 to October 2020, the Centers for Disease Control and Prevention was notified of 18 patients with M. chimaera infections who had undergone cardiothoracic surgeries at 2 hospitals in Kansas (14 patients) and California (4 patients); 17 had exposure to 3T HCDs. Whole-genome sequencing of the clinical and environmental isolates matched the global outbreak strain identified in 2015. METHODS: Investigations were conducted at each hospital to determine the cause of ongoing infections. Investigative methods included query of microbiologic records to identify additional cases, medical chart review, observations of operating room setup, HCD use and maintenance practices, and collection of HCD and environmental samples. RESULTS: Onsite observations identified deviations in the positioning and maintenance of the 3T HCDs from the US Food and Drug Administration (FDA) recommendations and the manufacturer's updated cleaning and disinfection protocols. Additionally, most 3T HCDs had not undergone the recommended vacuum and sealing upgrades by the manufacturer to decrease the dispersal of M. chimaera-containing aerosols into the operating room, despite hospital requests to the manufacturer. CONCLUSIONS: These findings highlight the need for continued awareness of the risk of M. chimaera infections associated with 3T HCDs, even if the devices are newly manufactured. Hospitals should maintain vigilance in adhering to FDA recommendations and the manufacturer's protocols and in identifying patients with potential M. chimaera infections with exposure to these devices.

Pulmonary nontuberculous mycobacterial infections and environmental factors: A review of the literature.

BACKGROUND: Pulmonary nontuberculous mycobacterial (pNTM) infection is mainly acquired through the inhalation of bioaerosols. Nevertheless, behavioural restrictions are rarely given by clinicians to susceptible populations, in part because the available guidelines for pNTM management emphasize more diagnosis and treatment than prevention. Aim of this review is to clarify if pNTM prevention should routinely include recommendations about risk reducing behaviors. METHODS: We used PubMed as biomedical database. We limited our search to the publication period 2000 to 2020 with selected keyword combinations including "nontuberculous mycobacteria", "water", "soil", and "exposure". Titles and abstract of selected articles were systematically screened. Articles were included in the analysis if they were published under free access through the digital library of the University of Brescia (Italy), and provided full text either in English, French, German or Italian. Articles were excluded if the topic was beyond the aim of our study. Finally, we selected 20 articles. RESULTS: Studies disagree in identifying the type of aerosol posing the highest risk for the development of pNTM infection. In the retrieved publications the colonization of household niches has been associated with a higher risk of pNTM disease, such as in the exposure to shower aerosols. Considering the non-household settings, the exposure to aerosols in indoor swimming and the higher soil exposure (>2 h/week) seem to correlate with a higher risk to develop pNTM disease. According to our findings, randomized behavioural intervention studies are missing. CONCLUSIONS: Stringent scientific evidence is missing to formulate recommendations on behavioural risk reduction for pNTM.

BCG turns 100: its nontraditional uses against viruses, cancer, and immunologic diseases.

First administered to a human subject as a tuberculosis (TB) vaccine on July 18, 1921, Bacillus Calmette-Guérin (BCG) has a long history of use for the prevention of TB and later the immunotherapy of bladder cancer. For TB prevention, BCG is given to infants born globally across over 180 countries and has been in use since the late 1920s. With about 352 million BCG doses procured annually and tens of billions of doses having been administered over the past century, it is estimated to be the most widely used vaccine in human history. While its roles for TB prevention and bladder cancer immunotherapy are widely appreciated, over the past century, BCG has been also studied for nontraditional purposes, which include (a) prevention of viral infections and nontuberculous mycobacterial infections, (b) cancer immunotherapy aside from bladder cancer, and (c) immunologic diseases, including multiple sclerosis, type 1 diabetes, and atopic diseases. The basis for these heterologous effects lies in the ability of BCG to alter immunologic set points via heterologous T cell immunity, as well as epigenetic and metabolomic changes in innate immune cells, a process called "trained immunity." In this Review, we provide an overview of what is known regarding the trained immunity mechanism of heterologous protection, and we describe the current knowledge base for these nontraditional uses of BCG.

Designing a multi-epitope vaccine against Mycobacteroides abscessus by pangenome-reverse vaccinology.

Mycobacteroides abscessus (Previously Mycobacterium abscessus) is an emerging microorganism of the newly defined genera Mycobacteroides that causes mainly skin and tissue diseases in humans. The recent availability of total 34 fully sequenced genomes of different strains belonging to this species has provided an opportunity to utilize this genomics data to gain novel insights and guide the development of specific antimicrobial therapies. In the present study, we collected collectively 34 complete genome sequences of M. abscessus from the NCBI GenBank database. Pangenome analysis was conducted on these genomes to understand the genetic diversity and to obtain proteins associated with its core genome. These core proteins were then subjected to various subtractive filters to identify potential antigenic targets that were subjected to multi-epitope vaccine design. Our analysis projected the open pangenome of M. abscessus containing 3443 core genes. After applying various stepwise filtration steps on the core proteins, a total of four potential antigenic targets were identified. Utilizing their constituent CD4 and CD8 T-cell epitopes, a multi-epitope based subunit vaccine was computationally designed. Sequence-based analysis as well as structural characterization revealed the immunological effectiveness of this designed vaccine. Further molecular docking, molecular dynamics simulation and binding free energy estimation with Toll-like receptor 2 indicated strong structural associations of the vaccine with the immune receptor. The promising results are encouraging and need to be validated by additional wet laboratory studies for confirmation.

Tap Water Avoidance Decreases Rates of Hospital-onset Pulmonary Nontuberculous Mycobacteria.

We analyzed the impact of a hospital tap water avoidance protocol on respiratory isolation of nontuberculous mycobacteria (NTM). After protocol implementation, hospital-onset episodes of respiratory NTM isolation on high-risk units decreased from 41.0 to 9.9 episodes per 10 000 patient-days (incidence rate ratio, 0.24; 95% confidence interval, .17-.34; P < .0001).

Spreading of a mycobacterial cell-surface lipid into host epithelial membranes promotes infectivity.

Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.

A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

Bacille Calmette Guérin (BCG) and new TB vaccines: Specific, cross-mycobacterial and off-target effects.

The Bacille Calmette Guérin (BCG) vaccine was developed over a century ago and has become one of the most used vaccines without undergoing a modern vaccine development life cycle. Despite this, the vaccine has protected many millions from severe and disseminated forms of tuberculosis (TB). In addition, BCG has cross-mycobacterial effects against non-tuberculous mycobacteria and off-target (also called non-specific or heterologous) effects against other infections and diseases. More recently, BCG's effects on innate immunity suggest it might improve the immune response against viral respiratory infections including SARS-CoV-2. New TB vaccines, developed over the last 30 years, show promise, particularly in prevention of progression to disease from TB infection in young adults. The role of BCG in the context of new TB vaccines remains uncertain as most participants included in trials have been previously BCG immunised. BCG replacement vaccines are in efficacy trials and these may also have off-target effects.

Sirtuin 3 is essential for host defense against Mycobacterium abscessus infection through regulation of mitochondrial homeostasis.

The global incidence of Mycobacterium abscessus (Mabc), a rapidly growing nontuberculous mycobacterial strain that causes treatment-refractory pulmonary diseases, is increasing. Despite this, the host factors that allow for protection against infection are largely unknown. In this study, we found that sirtuin 3 (SIRT3), a mitochondrial protein deacetylase, plays a critical role in host defense against Mabc infection. Mabc decreased SIRT3 and upregulated mitochondrial oxidative stress in macrophages. SIRT3 deficiency led to increased bacterial loads, histopathological, and mitochondrial damage, and pathological inflammation during Mabc infection. Administration of scavengers of mitochondrial reactive oxygen species significantly decreased the in vivo Mabc burden and excessive inflammation, and induced SIRT3 expression in infected lungs. Notably, SIRT3 agonist (resveratrol) significantly decreased Mabc growth and attenuated inflammation in mice and zebrafishes, indicating the key role for SIRT3 in metazoan host defense. Collectively, these data strongly suggest that SIRT3 is a host-directed therapeutic target against Mabc infection by controlling mitochondrial homeostasis.

DNA vaccination with the Mycobacterium marinum MMAR_4110 antigen inhibits reactivation of a latent mycobacterial infection in the adult Zebrafish.

BACKGROUND: Tuberculosis is a major challenge for health care, as options for its treatment and prevention are limited. Therefore, novel approaches, such as DNA vaccination, to both prevent primary infections and the reactivation of latent infections need to be developed. A Mycobacterium marinum infection in adult zebrafish (Danio rerio) recapitulates features of the human Mycobacterium tuberculosis infection, providing a convenient preclinical animal model for studying tuberculosis. METHODS: Hypoxic M. marinum cultures were produced with the Wayne model, and further reaerated to replicate the in vivo reactivation in vitro. Expression levels of M. marinum genes were studied with mRNA sequencing from exponentially growing bacteria, anaerobic cultures and at 2 and 12 h after reaeration. Seven reactivation-associated genes were selected for further studies, where their antigen potentiality as DNA-vaccines to prevent reactivation of a latent mycobacterial infection was investigated in the adult zebrafish model. The Mann-Whitney test was used to evaluate differences in bacterial counts between the groups. RESULTS: The mRNA sequencing data showed that, seven M. marinum genes, MMAR_0444, MMAR_0514, MMAR_0552, MMAR_0641, MMAR_1093, MMAR_4110 and MMAR_4524, were upregulated during reactivation when compared to both dormant and logarithmic growing bacteria. Four different MMAR_4110 antigens prevented the reactivation of a latent mycobacterial infection in the adult zebrafish. CONCLUSION: This study provides novel information about reactivation-related M. marinum genes. One of the antigens, MMAR_4110, inhibited the reactivation of a latent M. marinum infection in zebrafish, implicating that the characterized genes could be potential targets for further vaccine and drug development against mycobacterial diseases.

Current and future management of non-tuberculous mycobacterial pulmonary disease (NTM-PD) in the UK.

A rising number of non-tuberculous mycobacterial (NTM) isolates are being identified in UK clinical practice. There are many uncertainties around the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD), including its epidemiology, diagnosis, treatment and prevention. Regional variations in how patients with NTM-PD are managed reflects the lack of standardised pathways in the UK. Service optimisation and multidisciplinary working can improve the quality of care for patients with NTM-PD, including (1) better identification of patients at risk of NTM-PD and modification of risk factors where applicable; (2) standardisation of reference laboratory testing to offer clinicians access to accurate and prompt information on NTM species and drug sensitivities; (3) development of recognised specialist NTM nursing care; (4) standardisation of NTM-PD imaging strategies for monitoring of treatment and disease progression; (5) establishment of a hub-and-spoke model of care, including clear referral and management pathways, dedicated NTM-PD multidisciplinary teams, and long-term patient follow-up; (6) formation of clinical networks to link experts who manage diseases associated with NTM; (7) enabling patients to access relevant support groups that can provide information and support for their condition; and (8) development of NTM research groups to allow patient participation in clinical trials and to facilitate professional education.

Of tuberculosis and non-tuberculous mycobacterial infections - a comparative analysis of epidemiology, diagnosis and treatment.

Pulmonary diseases due to mycobacteria cause significant morbidity and mortality to human health. In addition to tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), recent epidemiological studies have shown the emergence of non-tuberculous mycobacteria (NTM) species in causing lung diseases in humans. Although more than 170 NTM species are present in various environmental niches, only a handful, primarily Mycobacterium avium complex and M. abscessus, have been implicated in pulmonary disease. While TB is transmitted through inhalation of aerosol droplets containing Mtb, generated by patients with symptomatic disease, NTM disease is mostly disseminated through aerosols originated from the environment. However, following inhalation, both Mtb and NTM are phagocytosed by alveolar macrophages in the lungs. Subsequently, various immune cells are recruited from the circulation to the site of infection, which leads to granuloma formation. Although the pathophysiology of TB and NTM diseases share several fundamental cellular and molecular events, the host-susceptibility to Mtb and NTM infections are different. Striking differences also exist in the disease presentation between TB and NTM cases. While NTM disease is primarily associated with bronchiectasis, this condition is rarely a predisposing factor for TB. Similarly, in Human Immunodeficiency Virus (HIV)-infected individuals, NTM disease presents as disseminated, extrapulmonary form rather than as a miliary, pulmonary disease, which is seen in Mtb infection. The diagnostic modalities for TB, including molecular diagnosis and drug-susceptibility testing (DST), are more advanced and possess a higher rate of sensitivity and specificity, compared to the tools available for NTM infections. In general, drug-sensitive TB is effectively treated with a standard multi-drug regimen containing well-defined first- and second-line antibiotics. However, the treatment of drug-resistant TB requires the additional, newer class of antibiotics in combination with or without the first and second-line drugs. In contrast, the NTM species display significant heterogeneity in their susceptibility to standard anti-TB drugs. Thus, the treatment for NTM diseases usually involves the use of macrolides and injectable aminoglycosides. Although well-established international guidelines are available, treatment of NTM disease is mostly empirical and not entirely successful. In general, the treatment duration is much longer for NTM diseases, compared to TB, and resection surgery of affected organ(s) is part of treatment for patients with NTM diseases that do not respond to the antibiotics treatment. Here, we discuss the epidemiology, diagnosis, and treatment modalities available for TB and NTM diseases of humans.

Lymphocyte responses to Mycobacterium tuberculosis and Mycobacterium bovis are similar between BCG-vaccinated patients with cystic fibrosis and healthy controls.

BACKGROUND: The low rate of nontuberculous mycobacteria (NTM) among Brazilian patients with cystic fibrosis (CF) may be due to cross-reactive Bacille Calmette-Guérin (BCG) vaccination. In the present pilot study, we aimed to compare the lymphocyte responses against Mycobacterium tuberculosis(Mtb) and Mycobacterium bovis (BCG) in BCG-vaccinated CF patients and healthy controls. METHODS: The lymphocyte responses of CF patients (n = 10) and healthy controls (n = 10) were assessed in terms of lymphocyte proliferation index (LPI), using flow cytometry. Median rates of each cell subtype - CD4, CD8, γδ T cells and CD19 (B) cells - were also determined. RESULTS: Median LPIs (CF vs. controls) were 22.9% vs. 13.0% (p = 0.481) and 23.1% vs. 17.6% (p = 0.481), upon stimulation with Mtb and BCG, respectively. Both groups had a predominant CD4 T cell response to Mtb (median rate = 82.5% vs. 79.7%; p = 0.796) and BCG (LPI = 84.3% vs. 83.0%; p = 0.853), which were significantly higher than the CD8, CD19 and γδ responses within both groups. CF patients tended to have a higher CD8 T cell response upon stimulation with the phytohemagglutinin mitogen than healthy controls (median rate = 42.8% vs. 31.7%, p = 0.075). CONCLUSION: The responses of BCG-vaccinated CF patients to Mtb and BCG are at least similar to those of healthy individuals. These are probably memory responses elicited by the BCG vaccination, which can cross-react with NTM and may explain the low frequency of NTM lung infection in our CF center.

Identification of Mycobacterium porcinum in patients with cystic Fibrosis: Pathogen or contaminant?

BACKGROUND: Mycobacterium porcinum is a non-tuberculous mycobacterium (NTM) identified in potable water. The identification and clinical impact of M. porcinum in patients with cystic fibrosis (CF) has not been described. In our institution, M. porcinum was isolated exclusively during hospitalization in a cluster of patients with CF. METHODS: Patients with CF who were hospitalized between September 2016 and September 2018 and could expectorate sputum were included, and samples were processed per institutional guidelines. Post-hospitalization and one-year clinical outcomes on those who isolated M. porcinum in respiratory cultures were reviewed. Whole genome sequencing was performed on M. porcinum isolates obtained from patients and environmental sources to identify source of acquisition. RESULTS: Review of 14 CF patients with 16 M. porcinum isolates revealed rapid time to culture positivity within 0.8 (0.04-8.0) days after admission. M. porcinum was isolated in teenagers and adults irrespective of baseline pulmonary function, body mass index, or CF genotype. Whole genome sequencing suggested all isolates belong to the same M. porcinum strain and confirmed the source of acquisition to the ice machine. Review of patients' clinical course, including three patients who underwent lung transplantation, suggested a pseudo-outbreak with minimal clinical impact. CONCLUSIONS: NTM, including M. porcinum, are ubiquitous in potable water and institutional water reservoirs. Our findings suggest M. porcinum is a transient colonizer rather than a pathogen. Challenges exist in discerning the role of NTM as a contributor of pulmonary morbidity in patients with CF, and adherence to established guidelines regarding NTM related pulmonary disease remains important.