Proteomics approach to understand reduced clearance of mycobacteria and high viral titers during HIV–mycobacteria co‐infection

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV‐infected population. These seemingly harmless non‐pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV‐associated complications. Although immune‐evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome‐enriched fractions from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) mono‐infected and HIV–M. bovis BCG co‐infected THP‐1 cells by LC‐MALDI‐MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co‐infection helped the survival of non‐pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co‐infection up‐regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co‐infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co‐infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.     

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease-infected animals

Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage^®) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml⁻¹) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low-level bacteraemia is associated with these infections in cattle. In a study of M. bovis-infected cattle (n = 41), the sensitivity of the Actiphage^® method was 95 % (95 % CI; 0.84–0.99) and specificity was 100 % (95% CI; 0.92–1). We further used Actiphage^® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne’s infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.     

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet‐induced obesity

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high‐fat diet‐induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil‐ and macrophage‐based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi‐infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high‐fat diet, toll‐like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow‐derived macrophages from obese, B. burgdorferi‐infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.     

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.     

Opposing effects on cardiac function by calorie restriction in different‐aged mice

Calorie restriction (CR) increases average and maximum lifespan and exhibits an apparent beneficial impact on age‐related diseases. Several studies have shown that CR initiated either in middle or old age could improve ischemic tolerance and rejuvenate the aging heart; however, the data are not uniform when initiated in young. The accurate time to initiate CR providing maximum benefits for cardiac remodeling and function during aging remains unclear. Thus, whether a similar degree of CR initiated in mice of different ages could exert a similar effect on myocardial protection was investigated in this study. C57BL/6 mice were subjected to a calorically restricted diet (40% less than the ad libitum diet) for 3 months initiated in 3, 12, and 19 months. It was found that CR significantly reversed the aging phenotypes of middle‐aged and old mice including cardiac remodeling (cardiomyocyte hypertrophy and cardiac fibrosis), inflammation, mitochondrial damage, telomere shortening, as well as senescence‐associated markers but accelerated in young mice. Furthermore, whole‐genome microarray demonstrated that the AMP‐activated protein kinase (AMPK)–Forkhead box subgroup ‘O’ (FOXO) pathway might be a major contributor to contrasting regulation by CR initiated in different ages; thus, increased autophagy was seen in middle‐aged and old mice but decreased in young mice. Together, the findings demonstrated promising myocardial protection by 40% CR should be initiated in middle or old age that may have vital implications for the practical nutritional regimen.     

Comparison of spleen transcriptomes of two wild rodent species reveals differences in the immune response against Borrelia afzelii

Different host species often differ considerably in susceptibility to a given pathogen, but the causes of such differences are rarely known. The natural hosts of the tick‐transmitted bacterium Borrelia afzelii, which is one of causative agents of Lyme borreliosis in humans, include a variety of small mammals like voles and mice. Previous studies have shown that B. afzelii‐infected bank voles (Myodes glareolus) have about ten times higher bacterial load than infected yellow‐necked mice (Apodemus flavicollis), indicating that these two species differ in resistance. In this study, we compared the immune response to B. afzelii infection in these host species by using RNA sequencing to quantify gene expression in spleen. Gene set enrichment analysis (GSEA) showed that several immune pathways were down‐regulated in infected animals in both bank voles and yellow‐necked mice. Moreover, IFNα response was up‐regulated in B. afzelii‐infected yellow‐necked mice, while IL6 signaling and the complement pathway were down‐regulated in infected bank voles; differences in regulation of these three pathways between bank voles and yellow‐necked mice could thus contribute to the difference in resistance to B. afzelii between the species. This study provides knowledge of gene expression induced by a zoonotic pathogen in its natural host, and possible species‐specific regulation of immune responses associated with resistance.     

Aging aggravates ischemic stroke‐induced brain damage in mice with chronic peripheral infection

Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all of which alter peripheral inflammatory processes with concomitant impact on stroke outcome. The majority of the stroke patients are elderly, but the impact of interactions between aging and inflammation on stroke remains unknown. We thus investigated the influence of age on the outcome of stroke in animals predisposed to systemic chronic infection. Th1‐polarized chronic systemic infection was induced in 18–22 month and 4‐month‐old C57BL/6j mice by administration of Trichuris muris (gut parasite). One month after infection, mice underwent permanent middle cerebral artery occlusion and infarct size, brain gliosis, and brain and plasma cytokine profiles were analyzed. Chronic infection increased the infarct size in aged but not in young mice at 24 h. Aged, ischemic mice showed altered plasma and brain cytokine responses, while the lesion size correlated with plasma prestroke levels of RANTES. Moreover, the old, infected mice exhibited significantly increased neutrophil recruitment and upregulation of both plasma interleukin‐17α and tumor necrosis factor‐α levels. Neither age nor infection status alone or in combination altered the ischemia‐induced brain microgliosis. Our results show that chronic peripheral infection in aged animals renders the brain more vulnerable to ischemic insults, possibly by increasing the invasion of neutrophils and altering the inflammation status in the blood and brain. Understanding the interactions between age and infections is crucial for developing a better therapeutic regimen for ischemic stroke and when modeling it as a disease of the elderly.     

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: Evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages

The intracellular growth kinetics ofMycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains ofMycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMMØ) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMMØ. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMMØ than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth ofM. tuberculosis H37Rv andM. avium was compared inside BMMØ from both theBcg ^s (C57BL/6) andBcg ^r (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained withM. tuberculosis-orM. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMMØ to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.     

Dietary rapamycin supplementation reverses age‐related vascular dysfunction and oxidative stress,while modulating nutrient‐sensing,cell cycle,and senescence pathways

Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.     

Association of Mycobacterium infections in patients with Mendelian susceptibility to mycobacterial disease with venous thromboembolism

An association between a hypercoagulable state and Mendelian susceptibility to mycobacterial disease (MSMD) has been established in a few studies; resultant thrombosis is considered rare. In a case‐control study, the prevalence of factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C mutations were investigated in mycobacterium‐infected patients. The study comprised 30 patients with mycobacterial infections (invasive, disseminated and/or recurrent infections with Bacille Calmette–Guerin or non‐tuberculosis mycobacteria and Mycobacterium Tuberculosis with positive results for acid‐fast bacilli and tuberculin skin tests) and 30 normal healthy controls. Forty female (66.7%) and 20 male subjects (33.3%) aged from 3 to 70 years were recruited into this study. Genotyping of targeted genes was performed by RT‐PCR and cytokine TNF‐α concentrations were quantified using a commercially available ELISA kit. Significant associations between mycobacterial infection and TNF‐α production after stimulating peripheral blood mononuclear cells with LPS alone and with IFN‐γ plus LPS were identified. Moreover, genotyping analysis in the studied population revealed a significant association between MTHFR c.677C>T (OR, 3.28; 95% CI, 1.35–7.92; P < 0.05), MTHFR c.1298A>C (OR, 2.33; 95% CI, 1.10–4.93; P < 0.05) and mycobacterial infection in affected patients, indicating susceptibility to venous thromboembolism according to previous studies. Additionally, mycobacterium‐infected patients had a significantly greater prevalence of MTHFR C677T and A1298C mutations than controls.     

Aging impairs peritoneal but not bone marrow‐derived macrophage phagocytosis

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age‐related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow‐derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow‐derived macrophages and bone marrow monocytes did not exhibit age‐related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age‐related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell‐derived IL‐10 was increased in resting and LPS‐activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue‐resident peritoneal macrophages, but not by bone marrow‐derived macrophages/monocytes, and suggest that age‐related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.     

Infection susceptibility and immune senescence with advancing age replicated in accelerated aging LmnaDhe mice

Aging confers increased susceptibility to common pathogens including influenza A virus. Despite shared vulnerability to infection with advancing age in humans and rodents, the relatively long time required for immune senescence to take hold practically restricts the use of naturally aged mice to investigate aging‐induced immunological shifts. Here, we show accelerated aging Lmna^Dhe mice with spontaneous mutation in the nuclear scaffolding protein, lamin A, replicate infection susceptibility, and substantial immune cell shifts that occur with advancing age. Naturally aged (≥20 month) and 2‐ to 3‐month‐old Lmna^Dhe mice share near identically increased influenza A susceptibility compared with age‐matched Lmna^WT control mice. Increased mortality and higher viral burden after influenza infection in Lmna^Dhe mice parallel reduced accumulation of lung alveolar macrophage cells, systemic expansion of immune suppressive Foxp3⁺ regulatory T cells, and skewed immune dominance among viral‐specific CD8⁺ T cells similar to the immunological phenotype of naturally aged mice. Thus, aging‐induced infection susceptibility and immune senescence are replicated in accelerated aging Lmna^Dhe mice.     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

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.     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

Background  

Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects.     

Life‐long caloric restriction reduces oxidative stress and preserves nitric oxide bioavailability and function in arteries of old mice

Aging impairs arterial function through oxidative stress and diminished nitric oxide (NO) bioavailability. Life‐long caloric restriction (CR) reduces oxidative stress, but its impact on arterial aging is incompletely understood. We tested the hypothesis that life‐long CR attenuates key features of arterial aging. Blood pressure, pulse wave velocity (PWV, arterial stiffness), carotid artery wall thickness and endothelium‐dependent dilation (EDD; endothelial function) were assessed in young (Y: 5–7 month), old ad libitum (Old AL: 30–31 month) and life‐long 40% CR old (30–31 month) B6D2F1 mice. Blood pressure was elevated with aging (P < 0.05) and was blunted by CR (P < 0.05 vs. Old AL). PWV was 27% greater in old vs. young AL‐fed mice (P < 0.05), and CR prevented this increase (P < 0.05 vs. Old AL). Carotid wall thickness was greater with age (P < 0.05), and CR reduced this by 30%. CR effects were associated with amelioration of age‐related changes in aortic collagen and elastin. Nitrotyrosine, a marker of cellular oxidative stress, and superoxide production were greater in old AL vs. young (P < 0.05) and CR attenuated these increase. Carotid artery EDD was impaired with age (P < 0.05); CR prevented this by enhancing NO and reducing superoxide‐dependent suppression of EDD (Both P < 0.05 vs. Old AL). This was associated with a blunted age‐related increase in NADPH oxidase activity and p67 expression, with increases in superoxide dismutase (SOD), total SOD, and catalase activities (All P < 0.05 Old CR vs. Old AL). Lastly, CR normalized age‐related changes in the critical nutrient‐sensing pathways SIRT‐1 and mTOR (P < 0.05 vs. Old AL). Our findings demonstrate that CR is an effective strategy for attenuation of arterial aging.     

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation,Structural Integrity,and Tolerance to Antibiotics

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.     

Tuberculosis: Smart manipulation of a lethal host

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global threat to human health. Development of drug resistance and co‐infection with HIV has increased the morbidity and mortality caused by TB. Macrophages serve as primary defense against microbial infections, including TB. Upon recognition and uptake of mycobacteria, macrophages initiate a series of events designed to lead to generation of effective immune responses and clearance of infection. However, pathogenic mycobacteria utilize multiple mechanisms for manipulating macrophage responses to protect itself from being killed and to survive within these cells that are designed to kill them. The outcomes of mycobacterial infection are determined by several host‐ and pathogen‐related factors. Significant advancements in understanding mycobacterial pathogenesis have been made in recent years. In this review, some of the important factors/mechanisms regulating mycobacterial survival inside macrophages are discussed.