Strains Of Mycobacterium Avium Research Articles

Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

Desiccation-Tolerance of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium abscessus and Mycobacterium chelonae.

Desiccation-tolerance of cells of four strains of Mycobacterium chimaera and individual strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium chelonae were measured by two methods. The survival of water-acclimated cells both in filter paper and on the surface of stainless-steel coupons were measured. In filter paper at 40% relative humidity at 25 °C, survival of patient isolates of M. avium and M. chimaera cells was 28% and 34% after 21 days of incubation, whereas it was 100% for the Sorin 3T isolate of M. chimaera. On stainless-steel biofilms after 42 days of incubation at 40% relative humidity at 25 °C, survival of water-acclimated cells of M. intracellulare was above 100%, while M. chelonae cells did not survive beyond 21 days, and survival of water-acclimated cells of M. avium and M. abscessus was 18% and 14%, respectively. On stainless-steel coupons, survival of patient and Sorin 3T isolates of M. chimaera was quite similar, specifically between 14% and 28% survival, after 42 days of incubation at 40% relative humidity at 25 °C. The experiments would support the hypothesis that some nontuberculous mycobacterial species are relatively desiccation-tolerant, whereas others are not. Further, long-term survival of the two M. chimaera strains is consistent with the presence of that species in Sorin 3T heater-coolers shipped throughout the world.

Evaluation of a virulent strain of Mycobacterium avium subsp. Paratuberculosis used as a heat-killed vaccine

Bovine paratuberculosis is one of the most important chronic infectious diseases in livestock. This disease is difficult to control because of its inefficient management (test and cull strategy and inadequate biosecurity). Thus, the development of an effective vaccine is essential. In this study, we evaluated a local virulent strain (6611) of Mycobacterium avium subsp. paratuberculosis as an inactivated vaccine in comparison with the Silirum vaccine in mouse model and cattle. Regarding the mice model, only the groups vaccinated with 6611 showed lower colony forming unit (CFU) counts with a lower lesion score in the liver in comparison to the control group at 6 and 12 weeks post-challenge (wpc). The immune response was predominantly humoral (IgG1), although both vaccinated groups presented a cellular response with IFNγ production as well, but the 6611 group had also significant production of IL-2, IL-6, IL-17a, TNF, and IL-10. In cattle, the 6611 vaccinated group was the only one that maintained significant antibody values at the end of the trial, with significant production of IgG2 and IFNγ. No PPDb reactor was detected in the vaccinated animals, according to the intradermal caudal fold tuberculin test. Our results indicate that the 6611 local strain protected mice from challenge with a virulent strain, by inducing a humoral and cellular immune response. In the bovine, the natural host, the evaluated vaccine also induced humoral and cellular immune responses, with higher levels of CD4 + CD25+ and CD8 + CD25+ T cells populations than the commercial vaccine. Despite the encouraging results obtained in this study, an experimental challenge trial in cattle is mandatory to evaluate the efficacy of our candidate vaccine in the main host.

Draft Genome Sequences of Two Bison-Type and Two Sheep-Type Strains of Mycobacterium avium subsp. paratuberculosis.

ABSTRACTGenome sequences of two type B and two type S strains of Mycobacterium avium subsp. paratuberculosis are presented. These strains were isolated in the United States from sheep, bison, and cattle suffering from Johne’s disease. These genomes will increase our understanding of the minor differences that exist among strains of this genetically stable subspecies.

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

Morphological and molecular characterisation of a new strain of Mycobacterium avium subsp. paratuberculosis in Swiss goats

Morphological and molecular characterisation of a new strain of Mycobacterium avium subsp. paratuberculosis in Swiss goats

Mycobacteria produce proteins involved in biofilm formation and growth-affecting processes.

The aim of this study was to determine the effect of mycobacterial proteins on mycobacterial biofilm formation and growth processes. We separated growth-affecting proteins (GEPs) from wild type of Mycobacterium bovis and ATCC strain of Mycobacterium avium subsp. avium. Our results showed that these mycobacteria-secreted GEPs are involved in biofilm formation, growth stimulatory, and inhibitory processes. Our findings suggest that GEP stimulated M. avium subsp. avium growth in vitro. Stimulation process was observed in mycobacteria affected with GEP extracted from M. avium subsp. avium. We found that both GEPs inhibited the growth of the M. bovis. Optical density measurement and visual analysis confirm that GEP plays an important role in biofilm formation process. Most of M. bovis GEP are associated with the type VII secretion and general secretion pathways. Our results contribute to a better understanding of the mechanisms underlying mycobacterial biofilm formation and growth-affecting processes and better characterization of mycobacterial proteins and their functions. It is noteworthy that this finding represents the first demonstration of GEP-mediated growth effects on a solid and liquid medium.

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.

Proteome Analysis of a M. avium Mutant Exposes a Novel Role of the Bifunctional Protein LysX in the Regulation of Metabolic Activity.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to β-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.

Use of Ultrasonication as a Rapid Pretreatment Method for MALDI-TOF MS of Mycobacterial Samples

Background: In Japan, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used as a simple and accurate method for mycobacterial identification, but existing pretreatment techniques are time-consuming and laborious.Objective: We characterized a new pretreatment technique that extracts proteins using ultrasonic disruption.Methods: We compared this new technique with the current pretreatment method and examined the usefulness of ultrasonication, including its use in mycobacterial inactivation. A total of 174 mycobacterial isolates were tested, including 50 Mycobacterium tuberculosis complex strains, 57 Mycobacterium avium strains, 55 Mycobacterium intracellulare strains, and 12 Mycobacterium kansasii strains. The ultrasonic pretreatment method was performed with or without heat-pretreatment at 95°C for 30 minutes, in parallel with the current conventional method. For all tested strains, the mycobacterial identification agreed when comparing the new and conventional methods.Results: Samples prepared by ultrasonication without heat pretreatment exhibited multiple significant differences when compared with samples prepared by ultrasonication with heat pretreatment or by conventional methods. However, most scores were over 2.0, and the lowest score exceeded 1.7. Furthermore, the new techniques could be performed in only 10 minutes for diagnosis. In addition, we confirmed that 1 minute of ultrasonic pretreatment yielded complete inactivation of M. tuberculosis complex strains.Conclusion: The more rapid technique of protein extraction/inactivation by ultrasonication without heat pretreatment is expected to be highly useful in clinical laboratory settings.

Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology.

Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology.

Synthesis and evaluation of antitubercular activity of fluorinated 5-aryl-4-(hetero)aryl substituted pyrimidines

Various 5-(fluoroaryl)-4-(hetero)aryl substituted pyrimidines have been synthesized based on the Suzuki cross-coupling and nucleophilic aromatic substitution of hydrogen (SNH) reactions starting from commercially available 5-bromopyrimidine and their antitubercular activity against Mycobacterium tuberculosis H37Rv has been explored. The outcome of the study disclose that, some of the compounds have showed promising activity in micromolar concentration against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant strains isolated from tuberculosis patients in Ural region (Russia). The data concerning the ‘structure–activity’ relationship for fluorinated compounds have been discussed.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25+ cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25+ cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

Boosting Effect of 2-Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium.

The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria.

Lack of the Transcription Factor Hypoxia-Inducible Factor 1α (HIF-1α) in Macrophages Accelerates the Necrosis of Mycobacterium avium-Induced Granulomas.

The establishment of mycobacterial infection is characterized by the formation of granulomas, which are well-organized aggregates of immune cells, namely, infected macrophages. The granuloma's main function is to constrain and prevent dissemination of the mycobacteria while focusing the immune response to a limited area. In some cases these lesions can grow progressively into large granulomas which can undergo central necrosis, thereby leading to their caseation. Macrophages are the most abundant cells present in the granuloma and are known to adapt under hypoxic conditions in order to avoid cell death. Our laboratory has developed a granuloma necrosis model that mimics the human pathology of Mycobacterium tuberculosis, using C57BL/6 mice infected intravenously with a low dose of a highly virulent strain of Mycobacterium avium. In this work, a mouse strain deleted of the hypoxia inducible factor 1α (HIF-1α) under the Cre-lox system regulated by the lysozyme M gene promoter was used to determine the relevance of HIF-1α in the caseation of granulomas. The genetic ablation of HIF-1α in the myeloid lineage causes the earlier emergence of granuloma necrosis and clearly induces an impairment of the resistance against M. avium infection coincident with the emergence of necrosis. The data provide evidence that granulomas become hypoxic before undergoing necrosis through the analysis of vascularization and quantification of HIF-1α in a necrotizing mouse model. Our results show that interfering with macrophage adaptation to hypoxia, such as through HIF-1α inactivation, accelerates granuloma necrosis.

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

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

Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum

Representative strains of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) grew at equal rates in laboratory medium at 21% (air) and 12% oxygen. Growth in 6% oxygen proceeded at a 1.4–1.8-fold lower rate. Colony formation was the same at 21% (air) and 6% oxygen. The MAIS strains survived rapid shifts from aerobic to anaerobic conditions as measured by two experimental approaches (Falkinham (1996) [1]). MAIS cells grown aerobically to log phase in broth were diluted, spread on agar medium, and incubated anaerobically for up to 20days at 37°C. Although no colonies formed anaerobically, upon transfer to aerobic conditions, greater than 25% of the colony forming units (CFU) survived after 20days of anaerobic incubation (Prince et al. (1989) [2]). MAIS cells grown in broth aerobically to log phase were sealed and vigorous agitation led to oxygen depletion (Wayne model). After 12days anaerobic incubation, M. avium and M. scrofulaceum survival were high (>50%), while M. intracellulare survival was lower (22%). M. avium cells shifted to anaerobiosis in broth had increased levels of glycine dehydrogenase and isocitrate lyase. Growth of MAIS strains at low oxygen levels and their survival following a rapid shift to anaerobiosis is consistent with their presence in environments with fluctuating oxygen levels.

Delivery of aerosolized liposomal amikacin as a novel approach for the treatment of nontuberculous mycobacteria in an experimental model of pulmonary infection.

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

Genetic characterization of German Mycobacterium avium strains isolated from different hosts and specimens by multilocus sequence typing

Infections caused by Mycobacterium avium and its subspecies are reported as emerging disease in many countries worldwide. In our study we applied the multilocus sequence typing technology to 98 German M. avium strains originating from different hosts and specimens to examine the degree of the genetic diversity. By MLST, 80% of strains were identified as subspecies ‘M. avium hominissuis’, and 20% as subspecies M. avium avium/M. avium silvaticum. Distinctly different MLST profiles were identified for both subspecies. Based on the analysis of 4 and 5 loci, 87 and 106 SNPs and 1 codon deletion could be detected, respectively, resulting in 40 different strain profiles. Twelve out of these have recently been described for strains coming from different countries, yet in our study, additional new strain profiles (n=28) were found. The high degree of diversity within ‘M. avium subsp. hominissuis’ as well as the relatedness of human, porcine and environmental strains could be confirmed by IS1245 RFLP fingerprinting. The detection of ISMav6 and hsp65 code 15 in one adult patient strain being positive for IS901, but displaying ‘M. avium subsp. hominissuis’ MLST profile revealed that PCR for detection of IS901 is not a definitive proof of M. avium subsp. avium/M. avium subsp. silvaticum.