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Non-tuberculous mycobacteria (NTM) are opportunistic pathogens ubiquitous in the environment. Mycobacteroides abscessus is a relatively new pathogen associated with underlying lung chronic pathologies, accounting for most of the pulmonary infections linked to the rapidly growing mycobacteria group. This includes chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. Patient outcomes from M. abscessus infections are poor due to complicated treatments and other factors. Intrinsic drug resistance plays an important role. The M. abscessus toolbox of resistance is varied leading to complex strategies for treatment. Mechanisms include waxy cell walls, drug export mechanisms, and acquired resistance. Many studies have also shown the impact of extracellular DNA found in the biofilm matrix during early infection and its possible advantage in pathogenicity. In this review, we discuss the current knowledge of early infection focusing on biofilm formation, an environmental strategy, and which treatments prevent its formation improving current antibiotic treatment outcomes in preliminary studies.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Objectives: Slow-growing nontuberculous mycobacteria (NTMs) are highly prevalent and routinely cause opportunistic intracellular infectious disease in immunocompromised hosts., Methods: The activity of the triple combination of antibiotics, clarithromycin (CLR), rifabutin (RFB), and clofazimine (CFZ), was evaluated and compared with the activity of single antibiotics as well as with double combinations in an in vitro biofilm assay and an in vivo murine model of Mycobacterium avium subsp. hominissuis ( M. avium ) lung infection., Results: Treatment of 1-week-old biofilms with the triple combination exerted the strongest effect of all (0.12 ± 0.5 × 10 7 CFU/mL) in reducing bacterial growth as compared to the untreated (5.20 ± 0.5 × 10 7 /mL) or any other combination (≥0.75 ± 0.6 × 10 7 /mL) by 7 days. The treatment of mice intranasally infected with M. avium with either CLR and CFZ or the triple combination provided the greatest reduction in CLR-sensitive M. avium bacterial counts in both the lung and spleen compared to any single antibiotic or remaining double combination by 4 weeks posttreatment. After 4 weeks of treatment with the triple combination, there were no resistant colonies detected in mice infected with a CLR-resistant strain. No clear relationships between treatment and spleen or lung organ weights were apparent after triple combination treatment., Conclusions: The biofilm assay data and mouse disease model efficacy results support the further investigation of the triple-antibiotic combination.

Tuberculosis, caused by Mycobacterium tuberculosis ( M. tb ), remains a significant global health challenge. The survival of M. tb in hostile extracellular and intracellular microenvironments is crucial for its pathogenicity. In this study, we discovered a Bacillus Calmette-Guérin (BCG) mutant B1033 that potentially affected mycobacterium pathogenicity. This mutant contained an insertion mutation gene, fad D33, which is involved in lipid metabolism; however, its direct role in regulating M. tb infection is not well understood. Here, we found that the absence of fad D 33 reduced BCG adhesion and invasion into human pulmonary alveolar epithelial cells and increased the permeability of the mycobacterial cell wall, allowing M. tb to survive in the low pH and membrane pressure extracellular microenvironment of the host cells. The absence of fad D 33 also inhibited the survival of BCG in macrophages by promoting the release of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumors necrosis factor-α, through the mitogen-activated protein kinase p38 signaling pathway. Overall, these findings provide new insights into M. tb mechanisms to evade host defenses and might contribute to identifying potential therapeutic and vaccine targets for tuberculosis prevention.

Mycobacterium avium subsp. hominissuis (MAH) is a common environmental bacterium that causes infection in immunocompromised patients such as those with HIV/AIDS, or patients with chronic lung disease such as cystic fibrosis. There are many strains of MAH with varying levels of virulence. Infection with MAH strains 100 and 104 has been associated with different immune responses in mice and outcome of the disease. While MAH 100 infection tends to be cleared from mice, MAH 104 is virulent and grows in host tissue. What is currently unknown are the mechanisms related to this difference in host defense and virulence. Our hypothesis is that differences in circulating innate lymphocytes response are associated with increased protection from infection. Innate lymphoid cells (ILC) are lymphoid cells with an important role in regulation of innate immune systems. ILCs can be categorized into three subpopulations ILC1, ILC2, and ILC3 based on their cytokine production and regulatory transcription factors. Investigation was carried out on how macrophage anti-MAH response change depending on activation by primary mouse lymphocytes activated with IL-12, IL-33, and IL-23, triggering differentiation into ILC-like subpopulations. Our results do not affirm the role of any one ILC subpopulation in macrophage anti-M. avium ability. Our findings instead support the conclusion that MAH infection of macrophages suppresses the stimulatory function of ILCs., (© 2023. The Author(s).)

Introduction: M. avium subsp. hominissuis (M. avium) is an intracellular, facultative bacterium known to colonize and infect the human host through ingestion or respiratory inhalation. The majority of pulmonary infections occur in association with pre- existing lung diseases, such as bronchiectasis, cystic fibrosis, or chronic obstructive pulmonary disease. M. avium is also acquired by the gastrointestinal route in immunocompromised individuals such as human immunodeficiency virus HIV-1 patients leading to disseminated disease. A hallmark of M. avium pulmonary infections is the ability of pathogen to form biofilms. In addition, M. avium can reside within granulomas of low oxygen and limited nutrient conditions while establishing a persistent niche through metabolic adaptations., Methods: Bacterial metabolic pathways used by M. avium within the host environment, however, are poorly understood. In this study, we analyzed M. avium proteome with a focus on core metabolic pathways expressed in the anaerobic, biofilm and aerobic conditions and that can be used by the pathogen to transition from one environment to another., Results: Overall, 3,715 common proteins were identified between all studied conditions and proteins with increased synthesis over the of the level of expression in aerobic condition were selected for analysis of in specific metabolic pathways. The data obtained from the M. avium proteome of biofilm phenotype demonstrates in enrichment of metabolic pathways involved in the fatty acid metabolism and biosynthesis of aromatic amino acid and cofactors. Here, we also highlight the importance of chloroalkene degradation pathway and anaerobic fermentationthat enhance during the transition of M. avium from aerobic to anaerobic condition. It was also found that the production of fumarate and succinate by MAV_0927, a conserved hypothetical protein, is essential for M. avium survival and for withstanding the stress condition in biofilm. In addition, the participation of regulatory genes/proteins such as the TetR family MAV_5151 appear to be necessary for M. avium survival under biofilm and anaerobic conditions., Conclusion: Collectively, our data reveal important core metabolic pathways that M. avium utilize under different stress conditions that allow the pathogen to survive in diverse host environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Abukhalid, Rojony, Danelishvili and Bermudez.)

Older individuals are at increased risk of developing severe respiratory infections. However, our understanding of the impact of aging on the respiratory tract remains limited as samples from healthy humans are challenging to obtain and results can be confounded by variables such as smoking and diet. Here, we carry out a comprehensive cross-sectional study (n = 34 adult, n = 49 aged) to define the consequences of aging on the lung using the rhesus macaque model. Pulmonary function testing establishes similar age and sex differences as humans. Additionally, we report increased abundance of alveolar and infiltrating macrophages and a concomitant decrease in T cells were in aged animals. scRNAseq reveals shifts from GRZMB to IFN expressing CD8 + T cells in the lungs. These data provide insight into age-related changes in the lungs' functional, microbial, and immunological landscape that explain increased prevalence and severity of respiratory diseases in the elderly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Virulent non-tuberculous Mycobacteria (NTMs) successfully reside and multiply within the phagosomes of phagocytic cells such as monocytes and macrophages. Macrophages play a very important role in the innate clearance of intracellular pathogens including NTMs. Attenuated Mycobacterium avium subsp. hominissuis 100 enters macrophages but is incapable of escaping these cells via canonical mycobacteria escape mechanisms. Alternatively, virulent Mycobacterium avium subsp. hominissuis 104 and Mycobacterium abscessus subsp. abscessus are able to modify macrophages to suit their growth, survival and ultimately escape from macrophages, while non-virulent Mycobacterium smegmatis is readily killed by macrophages. In this study we focused on early infection of macrophages with NTMs to determine the phenotypic response of macrophages, M1 or M2 differentiation, and phosphorylation alterations that can affect cellular response to invading bacteria. Our findings indicate that infection of the macrophage with MAH 100 and M. smegmatis favours the development of M1 macrophage, a pro-inflammatory phenotype associated with the killing of intracellular pathogens, while infection of the macrophage with MAH 104 and M. abscessus favoured the development of M2 macrophage, an anti-inflammatory phenotype associated with the healing process. Interference with the host post-translational mechanisms, such as protein phosphorylation, is a key strategy used by many intracellular bacterial pathogens to modulate macrophage phenotype and subvert macrophage function. By comparing protein phosphorylation patterns of infected macrophages, we observed that uptake of both MAH 100 and M. smegmatis resulted in MARCKS-related protein phosphorylation, which has been associated with macrophage activation. In contrast, in macrophages infected with MAH 104 and M. abscessus , methionine adenosyltransferase IIβ, an enzyme that catalyses the biosynthesis of S-adenosylmethionine, a methyl donor for DNA methylation. Inhibition of DNA methylation with 5-aza-2 deoxycytidine, significantly impaired the survival of MAH 104 in macrophages. Our findings suggest that the virulent MAH 104 and M. abscessus enhance its survival in the macrophage possibly through interference with the epigenome responses.

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

Non-tuberculosis mycobacteria (NTM) are ubiquitously found throughout the environment. NTM can cause respiratory infections in individuals with underlying lung conditions when inhaled, or systemic infections when ingested by patients with impaired immune systems. Current therapies can be ineffective at treating NTM respiratory infections, even after a long course or with multidrug treatment regimens. NTM, such as Mycobacterium avium subspecies hominissuis ( M. avium ), is an opportunistic pathogen that shares environments with ubiquitous free-living amoeba and other environmental hosts, possibly their evolutionary hosts. It is highly likely that interactions between M. avium and free-living amoeba have provided selective pressure on the bacteria to acquire survival mechanisms, which are also used against predation by macrophages. In macrophages, M. avium resides inside phagosomes and has been shown to exit it to infect other cells. M. avium's adaptation to the hostile intra-phagosomal environment is due to many virulence mechanisms. M. avium is able to switch the phenotype of the macrophage to be anti-inflammatory (M2). Here, we have focused on and discussed the bacterial defense mechanisms associated with the intra-phagosome phase of infection. M. avium possesses a plethora of antioxidant enzymes, including the superoxide dismutases, catalase and alkyl hydroperoxide reductase. When these defenses fail or are overtaken by robust oxidative burst, many other enzymes exist to repair damage incurred on M. avium proteins, including thioredoxin/thioredoxin reductase. Finally, M. avium has several oxidant sensors that induce transcription of antioxidant enzymes, oxidation repair enzymes and biofilm- promoting genes. These expressions induce physiological changes that allow M. avium to survive in the face of leukocyte-generated oxidative stress. We will discuss the strategies used by M. avium to infect human macrophages that evolved during its evolution from free-living amoeba. The more insight we gain about M. avium's mode of pathogenicity, the more targets we can have to direct new anti-virulence therapies toward.

Bacterial aggregation is a strategy employed by many pathogens to establish infection. Mycobacterium avium subsp. hominissuis (MAH) undergoes a phenotypic change, microaggregation, when exposed to the respiratory epithelium. We therefore compared how non-aggregated bacteria, or planktonic, and microaggregated MAH can establish lung infections by evaluating mucosal epithelial cell and phagocytic cell responses. It was determined that human mucosal lung epithelial cells recognition of MAH occurs through toll-like receptors 1 and 2. MAPK 1/3 is phosphorylated at 30 min post infection, and active at the transcriptional level 2 h post infection for both phenotypes. Microaggregate infected BEAS-2B cells up-regulated CCL5, IL-1β, and TNF-α cDNA, while planktonic infected cells only up-regulated IL-1β cDNA at 2 h post infection. Microaggregates are associated with increased uptake by macrophages after 1 h compared to planktonic bacteria (8.83% vs. 5.00%, P < 0.05). In addition, the microaggregate phenotype, when internalized by macrophages, had reduced growth compared to planktonic bacteria, which increased when the host cells were exposed to microaggregate supernatant, obtained from the incubation of MAH with HEp-2 cells. Moreover, microaggregate supernatant stimulated biofilm formation by planktonic and microaggregated bacteria. Microaggregate supernatant also induces the production of both pro- and anti-inflammatory cytokines, which was suppressed following MAH infection. The results suggest that epithelial recognition occurs during MAH infection, and the microaggregate phenotype stimulates an inflammatory response. The initial bacterial interaction with the mucosal epithelium and development of the microaggregate phenotype has a role in pathogenesis, allowing for more robust biofilm formation and infection establishment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cancer patients commonly develop infectious complications over the course of the disease. One thousand patients receiving treatment for an oncologic disease at a single veterinary teaching hospital were retrospectively reviewed for concurrent infections. A total of 153 confirmed bacterial infections were identified, 82 of which were abscesses or wounds, 13 of which were respiratory infections, 3 of which were ear infections, and 55 of which were urinary tract infections. It was observed that the majority of the infections were caused by bacteria that are normally associated with that specific site location. Escherichia coli was the most common pathogen linked to infections in general, but Staphylococcus pseudintermedius was a frequently identified pathogen associated with wound infections. The susceptibility to diverse antimicrobials varied with the site of infection. Eleven cases (7.1%) were caused by opportunistic infections of the site, and E. coli and Pseudomonas aeruginosa were the pathogens isolated. Those bacteria were resistant to many antibiotics but showed susceptibility to aminoglycosides, imipenem, quinolones, and polymyxin B. In conclusion, veterinary patients with cancer or those under treatment for tumors develop infections by commonly encountered bacteria in the different sites of the body, with a susceptibility to antibiotics that is not out of line from what is expected. A small subset of cases developed opportunistic infections, with microbes that were more resistant to many classes of antibiotics.

Johne's disease is a chronic and usually fatal enteric infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) and is responsible for hundreds of millions of dollars in losses for the agricultural industry. Natural infection typically begins with bacterial uptake and translocation through the epithelium of the small intestine, followed by ingestion by tissue macrophages and dissemination via the lymphatic or blood system throughout the body. To gain insights into the host responses and adaptation of MAP within phagocytic cells, we utilized the previously developed cell culture passage model, and mass spectrometric-based quantitative proteomic approach. Using the cell culture system, which mimics an in vivo interaction of MAP with intestinal epithelium and tissue macrophages, bacteria were passed through the bovine epithelial cells and, subsequently, used for macrophage infection (termed indirect infection), while uninfected cells and macrophage infection initiated with the culture grown bacteria (termed direct infection) served as controls. Approximately 3900 proteins were identified across all studied groups. The comparison within the subset of proteins that showed synthesis for more than two-fold in the direct infection over the uninfected control revealed an enrichment for the pro-inflammatory pathways such as the NF-κB and cytokine/chemokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. While these responses were absent in the indirect infection, cellular pathways such as cell cycle, healing, regulation of cell adhesion, ensemble of core extracellular matrix proteins, cell surface integrins and proteins mediating the integrin signaling were remarkably high within the indirect infection. In addition to global analysis of the macrophage proteome, we further validated the proteomics data and confirmed that MAP passage through epithelial cells modulates the expression and signaling of integrins in phagocytes. In this study, we demonstrate that predominant expression of integrins in the indirectly infected macrophages allows phagocytic cells to initiate stronger binding and efficient translocation through the endothelial cells, suggesting the important role of integrins in the spread of MAP infection.

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic intracellular pathogen causing infections in individuals with chronic lung conditions and patients with immune-deficient disorders. The treatment of MAH infections is prolonged and outcomes many times are suboptimal. The reason for the extended treatment is complex and reflects the inability of current antimicrobials to clear diverse phenotypes of MAH quickly, particularly, the subpopulation of susceptible but drug-tolerant bacilli where the persistent fitness to anti-MAH drugs is stimulated and enhanced by the host environmental stresses. In order to enhance the pathogen killing, we need to understand the fundamentals of persistence mechanism and conditions that can initiate the drug-tolerance phenotype in mycobacteria. MAH can influence the intracellular environment through manipulation of the metal concentrations in the phagosome of infected macrophages. While metals play important role and are crucial for many cellular functions, little is known how vacuole elements influence persistence state of MAH during intracellular growth. In this study, we utilized the in vitro model mimicking the metal concentrations and pH of MAH phagosome at 1 h and 24 h post-infection to distinguish if metals encountered in phagosome could act as a trigger factor for persistence phenotype. Antibiotic treatment of metal mix exposed MAH demonstrates that metals of the phagosome environment can enhance the persistence state, and greater number of tolerant bacteria is recovered from the 24 h metal mix when compared to the viable pathogen number in the 1 h metal mix and 7H9 growth control. In addition, bacterial phenotype induced by the 24 h metal mix increases MAH tolerance to macrophage killing in TNF-α and IFN-γ activated cells, confirming presence of persistent MAH in the 24 h metal mix condition. This work shows that the phagosome environment can promote persistence population in MAH, and that the population differs dependent on a concentration of metals.

The high prevalence of Johne's disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

Mycobacterium avium subsp. hominissuis (MAH) is a common intracellular pathogen that infects immunocompromised individuals and patients with pre-existing chronic lung diseases, such as cystic fibrosis, who develop chronic and persistent pulmonary infections. The metabolic remodeling of MAH in response to host environmental stresses or within biofilms formed in bronchial airways plays an important role in development of the persistence phenotype contributing to the pathogen's tolerance to antibiotic treatment. Recent studies suggest a direct relationship between bacterial metabolic state and antimicrobial susceptibility, and improved antibiotic efficacy has been associated with the enhanced metabolism in bacteria. In the current study, we tested approximately 200 exogenous carbon source-dependent metabolites and identified short-chain fatty acid (SCFA) substrates (propionic, butyric and caproic acids) that MAH can utilize in different physiological states. Selected SCFA enhanced MAH metabolic activity in planktonic and sessile states as well as in the static and established biofilms during nutrient-limited condition. The increased bacterial growth was observed in all conditions except in established biofilms. We also evaluated the influence of SCFA on MAH susceptibility to clinically used antibiotics in established biofilms and during infection of macrophages and found significant reduction in viable bacterial counts in vitro and in cultured macrophages, suggesting improved antibiotic effectiveness against persistent forms of MAH.

Lipoarabinomannan (LAM) and its biosynthetic precursors, phosphatidylinositol mannosides (PIMs) and lipomannan (LM) play important roles in the interactions of Mycobacterium tuberculosis with phagocytic cells and the modulation of the host immune response, but nothing is currently known of the impact of these cell envelope glycoconjugates on the physiology and pathogenicity of nontuberculous mycobacteria. We here report on the structures of Mycobacterium abscessus PIM, LM, and LAM. Intriguingly, these structures differ from those reported previously in other mycobacterial species in several respects, including the presence of a methyl substituent on one of the mannosyl residues of PIMs as well as the PIM anchor of LM and LAM, the size and branching pattern of the mannan backbone of LM and LAM, and the modification of the arabinan domain of LAM with both succinyl and acetyl substituents. Investigations into the biological significance of some of these structural oddities point to the important role of polysaccharide succinylation on the ability of M. abscessus to enter and survive inside human macrophages and epithelial cells and validate for the first time cell envelope polysaccharides as important modulators of the virulence of this emerging pathogen.

Mycobacterium avium is a human and animal pathogen that infects the host through the mucosal surfaces. Past work has demonstrated that the bacterium can interact with both the respiratory and gastrointestinal tracts. Those surfaces in the body are covered by a bilayer of a glycoprotein, mucin, which works as a physical barrier and a gel which contains antibacterial and antivirus properties. This current work shows that different strains of M . avium , in contrast to Escherichia coli , Pseudomonas aeruginosa , and Listeria monocytogenes , are not able to bind to mucins, MUC2 and MUC5b, the main mucins in the gastrointestinal and respiratory tracts, respectively. The lack of binding is due to the characteristics of the cell wall and is impaired by altering lipids, proteins, or glycolipids. M . avium , in contrast to E . coli , interacts with epithelial cells equally in the presence or absence of the mucin, suggesting that the cell wall of the pathogen can facilitate the bacterial movement through the mucin layer, towards the mucosal wall. In conclusion, the study has shown that M . avium can avoid the mucin barrier, which explains its ability to interact with the mucosal epithelium, even in absence of motion-related structures., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 Jessica Bechler and Luiz E. Bermudez.)

Diabetes emerged as major risk factor for severe acute respiratory syndrome (SARS) and adverse outcome in patients with the coronavirus disease 2019 (COVID-19). Nevertheless, the role of admission hyperglycemia in patients with COVID-19 has not been well-explored, yet. With this retrospective analysis, we report for the first time that hyperglycemia on day-1 is the best predictor of radiographic imaging of SARS-CoV2, regardless of the past medical history of diabetes. Admission hyperglycemia should not be overlooked, but adequately treated to improve the outcomes of COVID-19 patients with our without diabetes., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mycobacterium abscessus subsp. abscessus (MAB) is a clinically important nontuberculous mycobacterium (NTM) causing pulmonary infection in patients such as cystic fibrosis and bronchiectasis. MAB is naturally resistant to the majority of available antibiotics. In attempts to identify the fundamental response of MAB to aerobic, anaerobic, and biofilm conditions (as it is encountered in patients) and during exposure to antibiotics, we studied bacterial proteome using tandem mass tag mass spectrometry sequencing. Numerous de novo synthesized proteins belonging to diverse metabolic pathways were found in anaerobic and biofilm conditions, including glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, nitrogen metabolism, and glyoxylate and dicarboxylate metabolism. Upon exposure to amikacin and linezolid under stress environments, MAB displayed metabolic enrichment for glycerophospholipid metabolism and oxidative phosphorylation. By comparing proteomes of two significant NTMs, MAB and M. avium subsp. hominissuis , we found highly synthesized shared enzymes of oxidative phosphorylation, TCA cycle, glycolysis/gluconeogenesis, glyoxylate/dicarboxylate, nitrogen metabolism, peptidoglycan biosynthesis, and glycerophospholipid/glycerolipid metabolism. The activation of peptidoglycan and fatty acid biosynthesis pathways indicates the attempt of bacteria to modify the cell wall, influencing the susceptibility to antibiotics. This study establishes global changes in the synthesis of enzymes promoting the metabolic shift and enhancing the pathogen resistance to antibiotics within different environments., Competing Interests: The authors declare no conflict of interest.

Mycobacterium avium subsp. hominissuis (MAH) belongs to the clinically important non-tuberculous mycobacterial group that infects immunocompromised patients and individuals with underling lung conditions. The need for prolonged therapy is a major challenge of MAH treatment, influencing the development of persistent and drug-resistant infections. The reason why bactericidal drugs take several months to eliminate MAH is unknown. To investigate MAH proteome remodeling under aerobic, anaerobic and biofilm conditions (as it is encountered in patient lungs) and identify metabolic changes potentially associated with bacterial persistent state, we performed the relative protein quantitative analysis using Tandem Mass Tag Mass Spectrometry sequencing. MAH was exposed to amikacin (4 μg/ml) and clarithromycin (16 μg/ml) under aerobic, anaerobic or biofilm condition for 24 h and the response was compared with bacterial proteomics of the corresponding conditions. Overall, 4000 proteins were identified out of 5313 MAH proteome of across all experimental groups. Numerous sets of de novo synthesized proteins belonging to metabolic pathways not evidenced in aerobic condition were found commonly enriched in both anaerobic and biofilm conditions, including pantothenate and CoA biosynthesis, glycerolipid metabolism, nitrogen metabolism and chloroalkene degradation, known to be associated with bacterial tolerance in M. tuberculosis . The common pathways observed in anaerobic and biofilm conditions following drug treatments were peptidoglycan biosynthesis, glycerophospholipid metabolism and protein export. The LprB lipoprotein, highly synthesized in MAH biofilms during drug treatments and shown to be essential for M. tuberculosis virulence and survival in vivo, was selected and overexpressed in MAH. Results demonstrate that LprB is secreted in MAH biofilms and the overexpression clone is more tolerant to antimicrobials than the wild-type strain. Our study identified promising metabolic pathways that can be targeted to prevent the bacterial tolerance mechanism and, subsequently, reduce the length of MAH therapy., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Records of all Diagnostic laboratory submissions from 2012 to 2015 were examined and subjected to analysis according to species, location of infection, species of bacteria, and antibiotic resistance/susceptibility. A total of 23.8% of all culture isolates were Staphylococcus sp. Of those Staphylococcus, 43% were isolated from surgical site infections. Staphylococcus pseudintermedius accounted for approximately 28% of all staphylococcus cultures, while methicillin-resistant (MR) S. pseudintermedius accounted for 8% of all staphylococcus cultures. Environmental samples were also collected by swabbing surfaces in the intensive care unit (ICU) and anesthesia prep room at the OSU VTH. Isolated bacterial colonies were subjected to PCR for species identification and for the presence of the mecA gene. Ability of horizontal transfer in vitro of the mecA gene was evaluated by incubating the mecA positive bacterium, with the mecA negative bacterium, and then plated onto agar plates infused with known concentration of oxacillin. Colonies were then subjected to PCR for species and mecA identification. Horizontal transfer of the mecA gene was demonstrated and confirmed via PCR from MR S. epidermidis to MS S. pseudintermedius in an in vitro model that mimicked the veterinary hospital environment. Biofilms were established using four Staphylococcus species isolated from swabbing the Intensive Care Unit (ICU) and anesthesia prep room and were resistant when exposed to the current cleaning agent. Staphylococcus species makeup nearly ¼ of all infections at OSU VDL during the four years of the study, and MS S. pseudintermedius was shown to acquire the mecA gene from an environmental strain., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bovine tuberculosis (bTB) is a highly transmissible infection and remains of great concern as a zoonosis. The worldwide incidence of bTB is in rise, creating potential reservoir and increased infection risk for humans and animals. In attempts to identify novel surface antigens of Mycobacterium bovis as a proof-of-concept for potential inducers of protective immunity, we investigated surface proteome of M. bovis BCG strain that was cultured under the granuloma-like condition. We also demonstrated that the pathogen exposed to the biologically relevant environment has greater binding and invasion abilities to host cells than those of bacteria incubated under regular laboratory conditions. A total of 957 surface-exposed proteins were identified for BCG cultured under laboratory condition, whereas 1,097 proteins were expressed under the granuloma-like condition. The overexpression of Mb1524, Mb01&#95;03198, Mb1595&#95;p3681 (PhoU1 same as phoY1&#95;1), and Mb1595&#95;p0530 (HbhA) surface proteins in Mycobacterium smegmatis leads to increased binding and invasion to mucosal cells. We also examined the immunogenicity of purified recombinant proteins and tested M. smegmatis overexpressing these surface antigens for the induction of protective immunity in mice. Significantly high levels of specific IgA and IgG antibodies were observed in recombinant protein immunized groups by both inhalation and intraperitoneal (IP) routes, but only IP delivery induced high total IgA and IgG levels. We did not detect major differences in antibody levels in the M. smegmatis group that overexpressed surface antigens. In addition, the bacterial load was significantly reduced in the lungs of mice immunized with the combination of inhaled recombinant proteins. Our findings suggest that the activation of the mucosal immunity can lead to increased ability to confer protection upon M. bovis BCG infection.

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic pathogen that is ubiquitous in the environment and often isolated from faucets and showerheads. MAH mostly infects humans with an underlying disease, such as chronic pulmonary disorder, cystic fibrosis, or individuals that are immunocompromised. In recent years, MAH infections in patients without concurrent disease are increasing in prevalence as well. This pathogen is resistant to many antibiotics due to the impermeability of its envelope and due to the phenotypic resistance established within the host macrophages, making difficult to treat MAH infections. By screening a MAH transposon library for mutants that are susceptible to killing by reactive nitrogen intermediaries, we identified the MAV &#95;4644 ( MAV &#95;4644:Tn) gene knockout clone that was also significantly attenuated in growth within the host macrophages. Complementation of the mutant restored the wild-type phenotype. The MAV &#95;4644 gene encodes a dual-function protein with a putative pore-forming function and ADP-ribosyltransferase activity. Protein binding assay suggests that MAV&#95;4644 interacts with the host lysosomal peptidase cathepsin Z (CTSZ), a key regulator of the cell signaling and inflammation. Pathogenic mycobacteria have been shown to suppress the action of many cathepsins to establish their intracellular niche. Our results demonstrate that knocking-down the cathepsin Z in human macrophages rescues the attenuated phenotype of MAV &#95;4644:Tn clone. Although, the purified cathepsin Z by itself does not have any killing effect on MAH, it contributes to bacterial killing in the presence of the nitric oxide (NO). Our data suggest that the cathepsin Z is involved in early macrophage killing of MAH, and the virulence factor MAV&#95;4644 protects the pathogen from this process.

Aim: To investigate the formation of Mycobacterium avium membrane vesicles (MVs) within macrophage phagosomes., Materials & Methods: A phagosome model was utilized to characterize proteomics and lipidomics of MVs. A click chemistry-based enrichment assay was employed to examine the presence of MV proteins in the cytosol of host cells., Results: Exposure to metals at concentrations present in phagosomes triggers formation of bacterial MVs. Proteomics identified several virulence factors, including enzymes involved in the cell wall synthesis, lipid and fatty acid metabolism. Some of MV proteins were also identified in the cytosol of infected macrophages. MVs harbor dsDNA., Conclusion: M. avium produces MVs within phagosomes. MVs carry products with potential roles in modulation of host immune defenses and intracellular survival.

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo , treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted., (Copyright © 2018 American Society for Microbiology.)

Mycobacterium avium: subsp. hominissuis (MAH) is an opportunistic pathogen that commonly infects immunocompromised individuals. Recently, we described an invasive phenotypic change MAH undergoes when incubated with lung airway epithelial host cells for 24 h, which is accompanied with microaggregate formation in vitro. The microaggregate phenotype also resulted in higher colonization in the lungs of mice early during infection. Previously, we identified genes highly regulated during microaggregate formation and further characterized the function of two highly upregulated bacterial proteins, mycobacterial binding protein-1 (MBP-1) and mycobacterial inversion protein-1 (MIP-1), which were found to be involved in binding and invasion of the respiratory mucosa. While these studies are valuable in understanding the pathogenesis of MAH, they primarily investigated the bacteria during microaggregate infection without commenting on the differences in the host response to microaggregate and planktonic infection. The bacteria-host interaction between microaggregates and epithelial cells was examined in a variety of assays. Using a transwell polarized epithelial cell model, microaggregates translocated through the monolayer more efficiently than planktonic bacteria at set timepoints. In addition, during infection with microaggregate and planktonic bacteria, host phosphorylated proteins were identified revealing differences in immune response, glutathione synthesis, and apoptosis. The host immune response was further investigated by measuring pro-inflammatory cytokine secretion during microaggregate and planktonic infection of BEAS-2B bronchial epithelial cells. The epithelial cells secreted more CCL5 during infection with microaggregates suggesting that this chemokine may play an important role during microaggregate invasion. Subsequent experiments showed that microaggregates are formed more efficiently in the presence of CCL5, suggesting that MAH had evolved a strategy to use the host response in its benefit. Collectively, this study establishes the different nature of infection by planktonic bacteria and microaggregates.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium.

Mycobacterium avium subsp. hominissuis ( M. avium ) is a member of the non-tuberculous mycobacteria (NTM), and is a common cause of lung infection in patients with chronic NTM lung conditions. M. avium is an environmental bacterium believed to be transmitted from environmental sources. In this work we used a recently developed model in Caenorhabditis elegans to ask whether M. avium can be transmitted from host-to-host, and the bacterial genes associated with host colonization. Infection of C. elegans was carried out by placing the nematode in cultured with M. avium . Bacteria eliminated from the intestines of infected C. elegans were used to infect naïve nematodes. In parallel experiments, to identify colonization associated genes, a transposon library of M. avium was screened for the ability to bind to HEp-2 mucosal cells. Thirty clones were identified and five selected clones with impaired adherence to HEp-2 epithelial cells were used to infect C. elegans to determine the degree of colonization. It was determined that M. avium eliminated from infected C. elegans were able to colonize a naïve C. elegans with high efficiency. Thirty of the most adherence-deficient M. avium clones obtained from the HEp-2 cell screening were sequenced to identify the location of the transposon. Many of the genes associated with the bacterial cell wall synthesis were shown to be inactivated in the selected mutants. Five out of the 30 bacterial clones were then used to infect C. elegans . All five mutants had impaired ability to colonize C. elegans compared with the wild type bacteria (decrease of 1.5-2.0 logs, p < 0.05). The limitation of this work is that the model can be used for initial screening, but other more complex systems should be used to confirm the findings. C. elegans can be used as a model to test for M. avium adherence/colonization-associated virulence determinants. All the tested adherence-deficient clones that were examined had impaired ability to colonize the host C. elegans , and some can be potentially used to prevent colonization.

Mycobacterium avium subsp. hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptosis, exit apoptotic cells or via ingestion of infected apoptotic bodies subsequently infect neighboring macrophages. By evaluating 4,000 transposon mutants of M. avium in THP-1 cells, we identified clones that can trigger a new form of early host cell apoptosis, which is only observed upon entry into the "secondary-infected" macrophages. Inactivation of MAVA5&#95;06970 gene lead to significant attenuation in intracellular growth within macrophages and mice, and impaired M. avium to induce rapid apoptosis in the "secondary-infected" cells as measured by Annexin V-FITC detection assay. Complementation of MAVA5&#95;06970 gene corrected the attenuation as well as apoptotic phenotypes. The MAVA5&#95;06970 gene encodes for a secreted protein. Using the pull-down assay and then confirmed with the yeast two-hybrid screen, we found that MAVA5&#95;06970 effector interacts with the Secreted Phosphoprotein 1, the cytokine also known as Osteopontin. This interaction enhances the THP-1 cell apoptosis and, consequently, restricts the production of interleukin-12 that likely may limit the activation of the type I immunity pathway in vivo. This work identified a key virulence effector of M. avium that contributes to the cell-to-cell spread of the pathogen.

Mycobacterium avium subsp. hominissuis is associated with infection of immunocompromised individuals as well as patients with chronic lung disease. M. avium infects macrophages and actively interfere with the host killing machinery such as apoptosis and autophagy. Bacteria alter the normal endosomal trafficking, prevent the maturation of phagosomes and modify many signaling pathways inside of the macrophage by secreting effector molecules into the cytoplasm. To investigate whether M. avium needs to attach to the internal surface of the vacuole membrane before releasing efferent molecules, vacuole membrane proteins were purified and binding to the surface molecules present in intracellular bacteria was evaluated. The voltage-dependent anion channels (VDAC) were identified as components of M. avium vacuoles in macrophages. M. avium mmpL4 proteins were found to bind to VDAC-1 protein. The inactivation of VDAC-1 function either by pharmacological means or siRNA lead to significant decrease of M. avium survival. Although, we could not establish a role of VDAC channels in the transport of known secreted M. avium proteins, we demonstrated that the porin channels are associated with the export of bacterial cell wall lipids outside of vacuole. Suppression of the host phagosomal transport systems and the pathogen transporter may serve as therapeutic targets for infectious diseases.

Purpose: Mycobacterium avium subsp. hominissuis is a member of the M. avium complex, a heterogeneous group of bacteria that cause lung infection in immunocompetent patients or disseminated infection in patients with immunosuppression. The bacteria belonging to this complex have variable virulence, depending on the strain considered, and therefore a representative of the most common clinical phenotype was analysed., Methodology: The genomic sequences of four M. avium subsp. hominissuis isolates obtained from clinical specimens were completed. Mav101, Mav100 and MavA5 were isolated from the blood of patients with AIDS. MavA5 was disseminated from the lung, while Mav3388 was isolated from the lungs of a patient with chronic lung disease. The sequences were annotated using the published Mav104 genome as a blueprint. Functional and virulence analyses of the sequences were carried out. Mice studies comparing the virulence of the strains were performed., Results: Findings showed that while Mav101 was very similar to Mav104, there were numerous differences between Mav104 and the remaining strains at nucleotide and predicted protein levels. The presence of genes associated with biofilm formation and several known virulence-related genes were sometimes differentially present among the isolates, suggesting overlapping functions by different genetic determinants., Conclusions: The sequences provided important information about M. avium heterogenicity and evolution as a pathogen. The limitation is the lack of understanding on possible overlapping functions of genes/proteins.

Tuberculosis (TB) continues to be one of the most common bacterial infectious diseases and is the leading cause of death in many parts of the world. A major limitation of TB therapy is slow killing of the infecting organism, increasing the risk for the development of a tolerance phenotype and drug resistance. Studies indicate that Mycobacterium tuberculosis takes several days to be killed upon treatment with lethal concentrations of antibiotics both in vitro and in vivo To investigate how metabolic remodeling can enable transient bacterial survival during exposure to bactericidal concentrations of compounds, M. tuberculosis strain H37Rv was exposed to twice the MIC of isoniazid, rifampin, moxifloxacin, mefloquine, or bedaquiline for 24 h, 48 h, 4 days, and 6 days, and the bacterial proteomic response was analyzed using quantitative shotgun mass spectrometry. Numerous sets of de novo bacterial proteins were identified over the 6-day treatment. Network analysis and comparisons between the drug treatment groups revealed several shared sets of predominant proteins and enzymes simultaneously belonging to a number of diverse pathways. Overexpression of some of these proteins in the nonpathogenic Mycobacterium smegmatis extended bacterial survival upon exposure to bactericidal concentrations of antimicrobials, and inactivation of some proteins in M. tuberculosis prevented the pathogen from escaping the fast killing in vitro and in macrophages, as well. Our biology-driven approach identified promising bacterial metabolic pathways and enzymes that might be targeted by novel drugs to reduce the length of tuberculosis therapy., (Copyright © 2017 American Society for Microbiology.)

Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate., Importance: Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain significant quantities of eDNA in their biofilms. In some bacteria, eDNA is derived from dead cells, but that does not appear to be the case for all eDNA-containing organisms, including NTM. In this study, we found that eDNA export in NTM is conditionally dependent on the molecules to which the bacteria are exposed and that bicarbonate positively influences eDNA export. We also identified genes and proteins important for eDNA export, which begins to piece together a description of a mechanism for eDNA. Better understanding of eDNA export can give new targets for the development of antivirulence drugs, which are attractive because resistance to classical antibiotics is currently a significant problem., (Copyright © 2016 Rose and Bermudez.)

Background: Mycobacterium avium subsp. hominissuis is a common intracellular pathogen that infects patients with HIV/AIDS and cause lung infection in patients with underlying lung pathology. M.avium preferably infects macrophages and uses diverse mechanisms to alter phagosome maturation. Once in the macrophage, the pathogen can alter the host cellular defenses by secreting proteins into the cytosol of host cells, but despite considerable research, only a few secreted effector proteins have been identified. We hypothesized that the environmental cues inside the phagosome can trigger bacterial protein secretion. To identify M. avium secretome within the phagosome, we utilized a previously established in vitro system that mimics the metal ion concentrations and pH of the M. avium phagosome., Results: M. avium was exposed to phagosome metal concentrations for different time points and exported proteins were profiled and analyzed against bacterial proteins secreted in the culture medium. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 46 were unique to bacteria incubated in the metal mixture. Ten of potential effectors were selected and secretion of these proteins was monitored within M. avium infected mononuclear phagocytic cells using the beta-lactamase FRET-based reporter system. In addition, pull-down assay was performed for secreted calmodulin-like protein MAV&#95;1356 protein to evaluate for eukaryotic target. All examined M. avium proteins were secreted into the macrophage cytosol, and gene expression analysis suggested that the metal environment likely stimulates secretion of pre-made proteins. Further investigation of bacterial secreted MAV&#95;1356 protein, lead to the observation that the MAV&#95;1356 interacts with the host proteins Annexin A1 and Protein S100-A8., Conclusions: We established an in vitro system for the study if proteins secreted intracellularly, and revealed that the metal mixture mimicking the concentration of metals in the phagosome environment, triggers protein secretion.

Mycobacterium avium is an opportunistic bacterium associated with pathogenic behavior in both humans and animals. M. avium has evolved as a pathogen by having an environmental component in its life style. Prophages are the integrated viral forms in bacterium genome. They constitute about 10% - 20% of genome of many bacteria and they contribute to pathogenicity of microbes. We investigated whether the M. avium 104 genome contained prophages and evaluated the genes/proteins for putative functions. Three prophage genes were identified in the M. avium 104 database, and sequences were analyzed for specific motifs. The prophage sequences were then cloned in Mycobacterium smegmatis and the bacterial phenotype was evaluated in gain of function assays for environmental stresses, such as tolerance to extreme temperatures, UV light, biofilm formation and resistance to acid as well as macrophage survival. The results indicate that two of the prophage genes, MAV&#95;0696 and MAV&#95;2265, confer M. smegmatis with enhanced ability to produce biofilm. Using a Real-Time PCR, it was determined that MAV&#95;0696 and MAV&#95;2265 transcripts were upregulated upon biofilm formation by M. avium . The expression of MAV&#95;2265 gene was significantly higher at all selected time points. In addition, the expression of MAV&#95;2265 in M . smegmatis also led to significantly greater survival rate at pH 5.0 compared to the wild-type control. None of the other physical abilities were altered by overexpressing the prophage genes in M. smegmatis . In summary, we identified three prophage sequences in M. avium 104, from which two of them were found to be associated with biofilm formation and one with resistance to the acidic environment. Future studies will identify the mechanisms involved in the prophages function.

Alveolar macrophages are the main line of innate immune response against M. tuberculosis (Mtb) infection. However, these cells serve as the major intracellular niche for Mtb enhancing its survival, replication and, later on, cell-to-cell spread. Mtb-associated cytotoxicity of macrophages has been well documented, but limited information exists about mechanisms by which the pathogen induces cell necrosis. To identify virulence factors involved in the induction of necrosis, we screened 5,000 transposon mutants of Mtb for clones that failed to promote the host cell necrosis in a similar manner as the wild-type bacterium. Five Mtb mutants were identified as potential candidates inducing significantly lower levels of THP-1 cell damage in contrast to the H37Rv wild-type infection. Reduced levels of the cell damage by necrosis deficient mutants (NDMs) were also associated with delayed damage of mitochondrial membrane permeability when compared with the wild-type infection over time. Two knockout mutants of the Rv3873 gene, encoding a cell wall PPE68 protein of RD1 region, were identified out of 5 NDMs. Further investigation lead to the observation that PPE68 protein interacts and exports several unknown or known surface/secreted proteins, among them Rv2626c is associated with the host cell necrosis. When the Rv2626c gene is deleted from the genome of Mtb, the bacterium displays significantly less necrosis in THP-1 cells and, conversely, the overexpression of Rv2626c promotes the host cell necrosis at early time points of infections in contrast to the wild-type strain.

The nematode Caenorhabditis elegans has become a model system for studying the disease interaction between pathogens and the host. To determine whether the transparent nematode could serve as a useful model for Mycobacterium avium subspecies hominissuis (MAH) infection of the intestinal tract, worms were fed MAH and assayed for the effects of the bacterial infection on the worm. It was observed during feeding that viable MAH increases in the intestinal lumen in a time dependent manner. Ingestion of MAH was deemed non-toxic to worms as MAH-fed populations have similar survival curves to those fed E. coli strain OP50. Pulse-chase analysis using E. coli strain OP50 revealed that MAH colonize the intestinal tract, as viable MAH remain within the intestine after the assay. Visualization of intestinal MAH using histology and transmission electron microscopy demonstrates that MAH localizes to the intestinal lumen, as well as establishes direct contact with intestinal epithelium. Bacterial colonization appears to have a detrimental effect on the microvilli of the intestinal epithelial cells. The MAH ΔGPL/4B2 strain with a mutation in glycopeptidolipid production is deficient in binding to human epithelial cells (HEp-2), as well as deficient in its ability to bind to and colonize the intestinal tract of C. elegans as efficiently as wild-type MAH. These data indicate the C. elegans may serve as a useful model system for MAH pathogenesis and in determining the mechanisms used by MAH during infection and colonization of the intestinal epithelium., (© 2015. Published by The Company of Biologists Ltd.)

Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV&#95;2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV&#95;2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV&#95;2941 action and demonstrated that MAV&#95;2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV&#95;2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV&#95;2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV&#95;2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV&#95;2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacteria that infects immunocompromised humans. MAH cases are increasing in incidence, making it crucial to gain knowledge of the pathogenic mechanisms associated with the bacterium. MAH infects macrophages and after several days the infection triggers the phagocyte apoptosis. Many of the intracellular MAH escape the cell undergoing apoptosis leading to infection of neighboring macrophages. We screened a transposon bank of MAH mutants in U937 mononuclear phagocytes for the inability to escape macrophages undergoing apoptosis. Mutations in genes; MAV&#95;2235, MAV&#95;2120, MAV&#95;2410, and MAV&#95;4563 resulted in the inability of the bacteria to exit macrophages upon apoptosis. Complementation of the mutations corrected the phenotype either completely or partially. Testing for the ability of the mutants to survive in macrophages compared to the wild-type bacterium revealed that the mutant clones were not attenuated up to 4 days of infection. Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice. Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms. The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process. The study reveals a pathway of escape from macrophages, not shared with Mycobacterium tuberculosis.

Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection.

Western diets are high in fat and sucrose and can influence behavior and gut microbiota. There is growing evidence that altering the microbiome can influence the brain and behavior. This study was designed to determine whether diet-induced changes in the gut microbiota could contribute to alterations in anxiety, memory or cognitive flexibility. Two-month-old, male C57BL/6 mice were randomly assigned high-fat (42% fat, 43% carbohydrate (CHO), high-sucrose (12% fat, 70% CHO (primarily sucrose) or normal chow (13% kcal fat, 62% CHO) diets. Fecal microbiome analysis, step-down latency, novel object and novel location tasks were performed prior to and 2weeks after diet change. Water maze testing for long- and short-term memory and cognitive flexibility was conducted during weeks 5-6 post-diet change. Some similarities in alterations in the microbiome were seen in both the high-fat and high-sucrose diets (e.g., increased Clostridiales), as compared to the normal diet, but the percentage decreases in Bacteroidales were greater in the high-sucrose diet mice. Lactobacillales was only significantly increased in the high-sucrose diet group and Erysipelotrichales was only significantly affected by the high-fat diet. The high-sucrose diet group was significantly impaired in early development of a spatial bias for long-term memory, short-term memory and reversal training, compared to mice on normal diet. An increased focus on the former platform position was seen in both high-sucrose and high-fat groups during the reversal probe trials. There was no significant effect of diet on step-down, exploration or novel recognitions. Higher percentages of Clostridiales and lower expression of Bacteroidales in high-energy diets were related to the poorer cognitive flexibility in the reversal trials. These results suggest that changes in the microbiome may contribute to cognitive changes associated with eating a Western diet., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Urinary tract infection caused by Escherichia coli is a frequently observed condition both in humans and animals. Uropathogenic E. coli (UPEC) has been shown to have a pathogenicity island that enables them to infect the urinary tract. Because there is little information about the presence of UPEC-associated virulent genes in animal isolates this work was carried out with the intent to enhance the understanding about the strains of E.coli that cause infections in animals., Results: We screened 21 E. coli strains isolated causing urinary tract infection in domestic animals. Primers were designed to amplify urinary infection-associated genes. Nine genes, pap A, tcp C , fyu A, tpb A, Lma, hyl A, pic U, ton B, and flic C were then amplified and sequenced. Different from the human isolate CFT073, all the animals E. coli lack some of the pathogenesis-associated genes. Genes encoding for proteins used to scavenge iron appear not to be so necessary during animal infections as they are in human infection. In further investigation of phenotypic properties, it was observed that animal UPECs have significantly more impaired ability to form biofilms than human UPEC strain., Conclusions: This study identified significant differences between human and animal UPECs. This may have its roots in the fact that it is difficult to determine if an animal has symptoms. Future studies will focus on some of the observations., Competing Interests: Competing Interest The authors declare they have no competing interest.