Induction of killing of Mycobacterium avium subsp. hominissuis in macrophages by cytokine stimulated innate-like lymphoid cells is negatively affected by the pathogen.

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.

Challenging diagnosis and successful treatment of localised Mycobacterium avium subsp. hominissuis glossitis in a dog on long-term immunomodulatory therapy.

CASE HISTORY: A 3-year-old, intact female mixed-breed dog, weighing 7 kg, was presented with generalised swelling of the tongue, leading to impaired deglutition and episodes of dyspnoea. From the age of 2 years, the dog had been under immunosuppressive therapy due to atopic dermatitis. CLINICAL FINDINGS AND TREATMENT: Multiple nodular lesions at the apex of the tongue were noted as well as mandibular and retropharyngeal lymph node enlargement. Serum biochemistry results showed inflammatory changes. The results of several biopsies taken over 7 months indicated persistent pyogranulomatous and necrotising glossitis despite ongoing antimicrobial treatment, first with amoxicillin/clavulanic acid and then pradofloxacin. No foreign material, acid-fast bacteria or fungal hyphae were detected throughout. The final diagnosis of Mycobacterium avium subsp. hominissuis (Mah) was reached after PCR and bacterial culture were carried out on the third biopsy sample. Therapy was initiated with rifampicin, clarithromycin and doxycycline, leading to complete remission of the lesions. DIAGNOSIS: Severe chronic pyogranulomatous and necrotising glossitis associated with infection by Mah. CLINICAL RELEVANCE: This report describes challenges in the diagnosis and therapy of a localised Mah infection in an iatrogenically immunocompromised dog. Successful treatment was only achieved with a specific combination of antibiotics administered long-term. ABBREVIATIONS: AF: Acid-fast; ALP: Alkaline phosphatase; CT: Computed tomography; MAC: Mycobacterium avium complex; Mah: Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis Infection in a Domestic Rabbit, Germany.

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen present in soil and dust. We report M. avium subsp. hominissuis infection found in a domestic rabbit in Hannover, Germany, in May 2017.

Antibiotic Susceptibility and Genotyping of Mycobacterium avium Strains That Cause Pulmonary and Disseminated Infection.

Mycobacterium avium subsp. hominissuis mainly causes disseminated infection in immunocompromised hosts, such as individuals with human immunodeficiency virus (HIV) infection, and pulmonary infection in immunocompetent hosts. However, many aspects of the different types of M. avium subsp. hominissuis infection remain unclear. We examined the antibiotic susceptibilities and genotypes of M. avium subsp. hominissuis isolates from different hosts by performing drug susceptibility testing using eight antibiotics (clarithromycin, rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, and levofloxacin) and variable-number tandem-repeat (VNTR) typing analysis for 46 isolates from the sputa of HIV-negative patients with pulmonary M. avium subsp. hominissuis disease without previous antibiotic treatment and 30 isolates from the blood of HIV-positive patients with disseminated M. avium subsp. hominissuis disease. Interestingly, isolates from pulmonary M. avium subsp. hominissuis disease patients were more resistant to seven of the eight drugs, with the exception being rifampin, than isolates from HIV-positive patients. Moreover, VNTR typing analysis showed that the strains examined in this study were roughly classified into three clusters, and the genetic distance from reference strain 104 for isolates from pulmonary M. avium subsp. hominissuis disease patients was statistically significantly different from that for isolates from HIV-positive patients (P = 0.0018), suggesting that M. avium subsp. hominissuis strains that cause pulmonary and disseminated disease have genetically distinct features. Significant differences in susceptibility to seven of the eight drugs, with the exception being ethambutol, were noted among the three clusters. Collectively, these results suggest that an association between the type of M. avium subsp. hominissuis infection, drug susceptibility, and the VNTR genotype and the properties of M. avium subsp. hominissuis strains associated with the development of pulmonary disease are involved in higher levels of antibiotic resistance.

Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin.

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.

Response of the respiratory mucosal cells to mycobacterium avium subsp. Hominissuis microaggregate.

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.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Genotyping and strain distribution of Mycobacterium avium subspecies hominissuis isolated from humans and pigs in Belgium, 2011-2013.

Mycobacterium avium represents a health concern for both humans and pigs. The characterisation of its subspecies is an important step improving the understanding of the epidemiology and the control of this pathogen. Ninety-two human M. avium strains were selected for a retrospective study. Subspecies determination by rpoB sequencing and IS1245/IS901 analysis showed that 98.9% of Belgian human M. avium strains belong to the subspecies hominissuis (MAH). Some of these MAH strains present particular IS1245/IS901 profiles (absence of IS1245 and false IS901 detection provoked by the presence of ISMav6). In addition, 54 MAH strains isolated from submandibular lymph nodes of Belgian pigs with lymphadenitis were included in this study. Genotyping of human and porcine isolates was performed using multispacer sequence typing (MST). In total, 49 different MST types were identified among pig (n = 11) and human (n = 43) MA isolates, with only five shared by both hosts. Among these MST types, 34 were newly identified. Our findings demonstrate the extensive genetic diversity among MAH isolates. Some genotypes were more prevalent in human or pigs but no correlation was observed between MST type and place of residence or the farm of origin for human and porcine isolates respectively, suggesting an environmental source of infection.

Establishment of persistent enteric mycobacterial infection following streptomycin pre-treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a chronic gastrointestinal disease affecting ruminants. This disease remains widespread in part due to the limitations of available diagnostics and vaccines. A representative small animal model of disease could act as a valuable tool for studying its pathogenesis and to develop new methods for paratuberculosis control, but current models are lacking. Streptomycin pre-treatment can reduce colonization resistance and has previously been shown to improve enteric infection in a Salmonella model. Here, we investigated whether streptomycin pre-treatment of mice followed by MAP gavage could act as a model of paratuberculosis which mimics the natural route of infection and disease development in ruminants. The infection outcomes of MAP were compared to M. avium subsp. hominissuis (MAH), an environmental mycobacterium, and M. bovis and M. orygis, two tuberculous mycobacteria. Streptomycin pre-treatment was shown to consistently improve bacterial infection post-oral inoculation. This model led to chronic MAP infection of the intestines and mesenteric lymph nodes (MLNs) up to 24-weeks post-gavage, however there was no evidence of inflammation or disease. These infection outcomes were found to be specific to MAP. When the model was applied to a bacterium of lesser virulence MAH, the infection was comparatively transient. Mice infected with bacteria of greater virulence, M. bovis or M. orygis, developed chronic intestinal and MLN infection with pulmonary disease similar to zoonotic TB. Our findings suggest that a streptomycin pre-treatment mouse model could be applied to future studies to improve enteric infection with MAP and to investigate other modifications underlying MAP enteritis.

Ultrastructure of the Mycobacterium avium subsp. hominissuis Biofilm.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial pathogens responsible for chronic lung disease in humans. It is widely distributed in biofilms in natural and living environments. It is considered to be transmitted from the environment. Despite its importance in public health, the ultrastructure of the MAH biofilm remains largely unknown. The ultrastructure of a MAH-containing multispecies biofilm that formed naturally in a bathtub inlet was herein reported along with those of monoculture biofilms developed from microcolonies and pellicles formed in the laboratory. Scanning electron microscopy revealed an essentially multilayered bathtub biofilm that was packed with cocci and short and long rods connected by an extracellular matrix (ECM). Scattered mycobacterium-like rod-shaped cells were observed around biofilm chunks. The MAH monoculture biofilms that developed from microcolonies in vitro exhibited an assembly of flat layers covered with thin film-like ECM membranes. Numerous small bacterial cells (0.76±0.19| |µm in length) were observed, but not embedded in ECM. A glycopeptidolipid-deficient strain did not develop the layered ECM membrane architecture, suggesting its essential role in the development of biofilms. The pellicle biofilm also consisted of flat layered cells covered with an ECM membrane and small cells. MAH alone generated a flat layered biofilm covered with an ECM membrane. This unique structure may be suitable for resistance to water flow and disinfectants and the exclusion of fast-growing competitors, and small cells in biofilms may contribute to the formation and transmission of bioaerosols.

Evaluation of Three Commercial Interferon-γ Assays in a Bovine Tuberculosis Free Population.

The interferon-γ assay has been used worldwide as an ancillary test for the diagnosis of bovine tuberculosis (bTB). This study aimed to describe, based on the bTB-free status in Switzerland, the difference of applying a more stringent cutoff point of 0.05 compared with 0.1 for bTB surveillance. Moreover, the effect of time between blood collection and stimulation, culture results, optical density values, and the influence of testing different breeds were evaluated. Blood samples from a total of 118 healthy cows older than 6 months were tested with three commercial interferon-gamma assays. To confirm the bTB-free status of the tested animals and to investigate potential cross-reactions with nontuberculous mycobacteria, pulmonary and abdominal lymph nodes in addition to ileal mucosa from each cattle were used for the detection of viable Mycobacteria spp. by specific culture. Significant differences regarding the proportion of false-positive results between the two Bovigam tests and between Bovigam 2G and ID Screen were found. Samples analyzed with Bovigam 2G were 2.5 [95% confidence interval (CI) 1.6-3.9] times more likely to yield a false-positive test result than samples analyzed with Bovigam TB. Similarly, the odds ratio (OR) for testing samples false-positive with ID Screen compared with Bovigam TB was 1.9 (95% CI 1.21-2.9). The OR for testing false-positive with ID Screen compared with Bovigam 2G was less to equally likely with an OR of 0.75 (95% CI 0.5-1.1). When using a cutoff of 0.05 instead of 0.1, the OR for a false-positive test result was 2.2 (95% CI 1.6-3.1). Samples tested after 6 h compared with a delayed stimulation time of 22-24 h were more likely to yield a false-positive test result with an OR of 3.9 (95% CI 2.7-5.6). In conclusion, applying a more stringent cutoff of 0.05 with the Bovigam 2G kit generates a questionable high number of false-positive results of one of three tested animals. Furthermore, specific breeds might show an increased risk to result false-positive in the Bovigam 2G and the ID Screen assays.

Interferon-γ Response of Mycobacterium avium subsp. paratuberculosis Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

Despite its potential for early diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of Mycobacterium bovis-specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or Mycobacterium avium subsp. hominissuis (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Mycobacterium avium Complex-specific antibody and PPDj-induced IFN-γ responses were monitored in vivo. Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of Mycobacterium bovis was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with Mycobacterium bovis peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.

Exposure of Mycobacterium avium subsp. homonissuis to Metal Concentrations of the Phagosome Environment Enhances the Selection of Persistent Subpopulation to Antibiotic Treatment.

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.

Isolation, Molecular Typing, and Antibiotic Susceptibility Testing of Mycobacterium avium Subspecies hominissuis From a Dog With Generalized Mycobacteriosis.

Mycobacterium avium-intracellulare complex infections are becoming an increasing concern in veterinary medicine because they affect livestock, wildlife, and companion animals. Here we describe the isolation, molecular typing, and antibiotic susceptibility testing of the causative agent of a rare case of generalized mycobacteriosis in a crossbred dog. Mycobacterial colonies were isolated from a popliteal lymph node aspirate sample and molecular typed by SNPs typing of the genes gyrB and rpsA, the 3' region of the hsp65 gene and the internal transcribed spacer (ITS), and MIRU-VNTR analysis. Colonies were also tested in vitro against the macrolide clarithromycin and other drugs, using a resazurin microdilution assay, in order to provide the most appropriate treatment for the dog. Results from SNPs typing of gyrB and ITS, as well as from MIRU-VNTR analysis suggested the isolation of a single strain of M. avium subsp. hominissuis (Mah). On the other hand, SNP typing of rpsA revealed DNA polymorphisms that led colonies to cluster into two groups. The presence of two distinct strains of Mah has been assumed. All colonies, regardless of the nucleotide sequence of rpsA, were found to be sensitive to all of the drugs tested except for ethambutol. Although the therapy administered was adequate, the dog's overall clinical status worsened progressively and the animal died 8 months later. In conclusion, we report on the isolation of Mah from a dog with generalized mycobacteriosis.

MAV_4644 Interaction with the Host Cathepsin Z Protects Mycobacterium avium subsp. hominissuis from Rapid Macrophage Killing.

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_4644 (MAV_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_4644 gene encodes a dual-function protein with a putative pore-forming function and ADP-ribosyltransferase activity. Protein binding assay suggests that MAV_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_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_4644 protects the pathogen from this process.

Genotyping of swine Mycobacterium avium subsp. hominissuis isolates from Kyushu, Japan.

The incidence of diseases caused by nontuberculous mycobacteria (NTM) is increasing annually worldwide, including Japan. Mycobacterium avium subsp. hoiminissuis (MAH) is one of the most common NTM species responsible for chronic lung diseases in animals and humans. In the current study, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing was employed to characterize the genetic diversity of swine MAH isolates from Kyushu, Japan. In total, 309 isolates were obtained from the lymph nodes of 107 pigs not displaying any clinical signs of disease, of which 307 were identified as MAH, comprising 173 strains. Based on eight established MIRU-VNTR loci, the MAH strains represented 50 genotypes constituting three lineages, and 29 had not been described in the Mac French National Institute for Agricultural Research Nouzilly MIRU-VNTR (Mac-INMV) database. MAH was the dominant M. avium complex (MAC) in pigs from Kyushu, and there was high genetic diversity among genotype profiles of MAH from Kyushu. We identified three predominant genotype profiles in the tested area sharing high relatedness with genotype profiles of strains isolated in European countries. MAH was the most common NTM in pigs from Kyushu and exhibited high diversity, with new strain-derived genotypes.

Genotyping and clarithromycin susceptibility testing of Mycobacterium avium subsp. hominissuis isolated in Tuscany, Italy.

Mycobacterium avium subsp. hominissuis (MAH) is a major cause of nontuberculous mycobacteria infection and the incidence of MAH infections is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity and the susceptibility to clarithromycin of a collection of 71 MAH human strains isolated in the last seven years. The VNTR analysis, revealing 16 unique patterns and 8 clusters including a total of 55 isolates, showed that most MAH isolates displayed a close genetic relationship, indicating that the MAH genotypes are quite homogeneous in our geographical area. Clarithromycin showed strong antimicrobial activity against MAH isolates, as indicated by the high proportion (94.4%) of susceptible strains. No association between specific VNTR patterns and the clinical features or the MIC of clarithromycin was found.

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model.

Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

Molecular Characterization of Mycobacterium avium subsp. hominissuis of Two Groups of Lymph Nodes, Being Intradermal Tuberculin or Interferon-Gamma Test Positive and Negative, Isolated from Swiss Cattle at Slaughter.

Mycobacterium avium subsp. hominissuis (MAH) is an important zoonotic pathogen with raising global health concerns. In humans, MAH is one of the most widespread non-tuberculous mycobacterial species responsible for lung disease. In animals, MAH is frequently isolated from pigs; however, it is also an opportunistic pathogen for other mammals including cattle. To elucidate the genetic diversity of MAH in cattle, a molecular characterization of isolates (n = 26) derived from lymph nodes was performed. Fourteen isolates originated from slaughtered cattle with visible altered lymph nodes at meat inspection, whereas 12 isolates were from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Variable number of tandem repeat (VNTR) analysis was performed at 20 loci to examine genetic differences of isolates and to compare to previously reported VNTR data of human isolates from different countries. Genetic elements IS901, IS1245, IS1311, LSPA17, ITS1 sequevar, and hsp65 code were determined. Interestingly, two bovine MAH isolates harbored ISMav6 and hsp65 code 15, which so far has only been observed in human isolates. We supposed that VNTR data of Swiss samples would show clustering with European samples. Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated a specific cluster of MAH isolates obtained from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Comparing Swiss isolates with isolates from different other countries, no geographical clustering was observed; however, four Swiss isolates had an identical VNTR profile as human isolates from the Netherlands, the United States, and Japan. These findings indicate a possible public health issue.

Induction of killing of Mycobacterium avium subsp. hominissuis in macrophages by cytokine stimulated innate-like lymphoid cells is negatively affected by the pathogen / Inducción de la destrucción de Mycobacterium avium subsp. hominissuis en macrófagos por células linfoides de tipo innato estimuladas por citoquinas se ve afectado negativamente por el patógeno

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.(AU)