River Sediments Downstream of Villages in a Karstic Watershed Exhibited Increased Numbers and Higher Diversity of Nontuberculous Mycobacteria.

The impact of residential villages on the nontuberculous mycobacteria (NTM) in streams flowing through them has not been studied in detail. Water and sediments of streams are highly susceptible to anthropogenic inputs such as surface water flows. This study investigated the impact of seven residential villages in a karst watershed on the prevalence and species spectrum of NTM in water and sediments. Higher NTM species diversity (i.e., 19 out of 28 detected) was recorded downstream of the villages and wastewater treatment plants (WWTPs) compared to sampling sites upstream (i.e., 5). Significantly, higher Zn and lower silicon concentrations were detected in sediments inside the village and downstream of the WWTP's effluents. Higher phosphorus concentration in sediment was downstream of WWTPs compared to other sampling sites. The effluent from the WWTPs had a substantial impact on water quality parameters with significant increases in total phosphorus, anions (Cl-and N-NH3-), and cations (Na+ and K+). The results provide insights into NTM numbers and species diversity distribution in a karst watershed and the impact of urban areas. Although in this report the focus is on the NTM, it is likely that other water and sediment microbes will be influenced as well.

Distinguishing Invasive from Chronic Pulmonary Infections: Host Pentraxin 3 and Fungal Siderophores in Bronchoalveolar Lavage Fluids.

The multiple forms of pulmonary aspergillosis caused by Aspergillus species are the most common respiratory mycoses. Although invasive, the analysis of diagnostic biomarkers in bronchoalveolar lavage fluid (BALF) is a clinical standard for diagnosing these conditions. The BALF samples from 22 patients with proven or probable aspergillosis were assayed for human pentraxin 3 (Ptx3), fungal ferricrocin (Fc), and triacetylfusarinine C (TafC) in a retrospective study. The infected group included patients with invasive pulmonary aspergillosis (IPA) and chronic aspergillosis (CPA). The BALF data were compared to a control cohort of 67 patients with invasive pulmonary mucormycosis (IPM), non-Aspergillus colonization, or bacterial infections. The median Ptx3 concentrations in patients with and without aspergillosis were 4545.5 and 242.0 pg/mL, respectively (95% CI, p < 0.05). The optimum Ptx3 cutoff for IPA was 2545 pg/mL, giving a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100, 98, 95, and 100%, respectively. The median Ptx3 concentration for IPM was high at 4326 pg/mL. Pentraxin 3 assay alone can distinguish IPA from CPA and invasive fungal disease from colonization. Combining Ptx3 and TafC assays enabled the diagnostic discrimination of IPM and IPA, giving a specificity and PPV of 100%.

Nontuberculous Mycobacteria: Ecology and Impact on Animal and Human Health.

Nontuberculous mycobacteria (NTM) represent an important group of environmentally saprophytic and potentially pathogenic bacteria that can cause serious mycobacterioses in humans and animals. The sources of infections often remain undetected except for soil- or water-borne, water-washed, water-based, or water-related infections caused by groups of the Mycobacterium (M.) avium complex; M. fortuitum; and other NTM species, including M. marinum infection, known as fish tank granuloma, and M. ulcerans infection, which is described as a Buruli ulcer. NTM could be considered as water-borne, air-borne, and soil-borne pathogens (sapronoses). A lot of clinically relevant NTM species could be considered due to the enormity of published data on permanent, periodic, transient, and incidental sapronoses. Interest is currently increasing in mycobacterioses diagnosed in humans and husbandry animals (esp. pigs) caused by NTM species present in peat bogs, potting soil, garden peat, bat and bird guano, and other matrices used as garden fertilizers. NTM are present in dust particles and in water aerosols, which represent certain factors during aerogenous infection in immunosuppressed host organisms during hospitalization, speleotherapy, and leisure activities. For this Special Issue, a collection of articles providing a current view of the research on NTM-including the clinical relevance, therapy, prevention of mycobacterioses, epidemiology, and ecology-are addressed.

Nontuberculous Mycobacteria as Sapronoses: A Review.

Mycobacteria are a unique group of microorganisms. They are characterised by exceptional adaptability and durability. They are capable of colonisation and survival even in very unfavourable conditions. In addition to the well-known obligate human pathogens, Mycobacterium tuberculosis and M. leprae, more than 200 other species have been described. Most of them form a natural part of the microflora of the external environment and thrive in aquatic and soil environments especially. For many of the mycobacterial species associated with human disease, their natural source has not yet been identified. From an ecological point of view, mycobacteria are saprophytes, and their application in human and animal diseases is opportunistic. Most cases of human disease from saprophytic mycobacteria occur in immunocompromised individuals. This adaptability and resilience to environmental pressures makes treatment of mycobacterial diseases (most often sapronoses and less often zoonoses) and permanent eradication of mycobacteria from the environment very difficult. Saprophytic mycobacterial diseases (sapronoses) are chronic and recurrent due to the fact of repeated endogenous or exogenous re-exposure. Therefore, knowledge regarding their occurrence in soil and dust would aid in the prevention of saprophytic mycobacterioses. In conjunction, their presence and ecological significance in the environment can be revealed.

Evaluation and comparison of antibiotic susceptibility profiles of Streptomyces spp. from clinical specimens revealed common and region-dependent resistance patterns.

Notwithstanding the fact that streptomycetes are overlooked in clinical laboratories, studies describing their occurrence in disease and potential pathogenicity are emerging. Information on their species diversity in clinical specimens, aetiology and appropriate therapeutic treatment is scarce. We identified and evaluated the antibiotic susceptibility profile of 84 Streptomyces clinical isolates from the Czech Republic. In the absence of appropriate disk diffusion (DD) breakpoints for antibiotic susceptibility testing (AST) of Streptomyces spp., we determined DD breakpoints by correlation with the broth microdilution method and by the distribution of zone diameters among isolates. Correlation accuracy was high for 9 antibiotics, leading to the establishment of the most valid DD breakpoints for Streptomyces antibiotic susceptibility evaluation so far. Clinical strains belonged to 17 different phylotypes dominated by a cluster of strains sharing the same percentage of 16S rRNA gene sequence identity with more than one species (S. albidoflavus group, S. hydrogenans, S. resistomycificus, S. griseochromogenes; 70% of isolates). AST results showed that Streptomyces exhibited intrinsic resistance to penicillin, general susceptibility to amikacin, gentamycin, vancomycin and linezolid, and high percentage of susceptibility to tetracyclines and clarithromycin. For the remaining antibiotics, AST showed inter- and intra-species variations when compared to available literature (erythromycin, trimethoprim-sulfamethoxazole), indicating a region-dependent rather than species-specific patterns.

Nontuberculous Mycobacteria Prevalence in Aerosol and Spiders' Webs in Karst Caves: Low Risk for Speleotherapy.

A total of 152 aerosol and spider web samples were collected: 96 spider's webs in karst areas in 4 European countries (Czech Republic, France, Italy, and Slovakia), specifically from the surface environment (n = 44), photic zones of caves (n = 26), and inside (aphotic zones) of caves (n = 26), 56 Particulate Matter (PM) samples from the Sloupsko-Sosuvsky Cave System (speleotherapy facility; n = 21) and from aerosol collected from the nearby city of Brno (n = 35) in the Czech Republic. Nontuberculous mycobacteria (NTM) were isolated from 13 (13.5%) spider's webs: 5 isolates of saprophytic NTM (Mycobacterium gordonae, M. kumamotonense, M. terrae, and M. terrae complex) and 6 isolates of potentially pathogenic NTM (M. avium ssp. hominissuis, M. fortuitum, M. intracellulare, M. peregrinum and M. triplex). NTM were not isolated from PM collected from cave with the speleotherapy facility although mycobacterial DNA was detected in 8 (14.3%) samples. Temperature (8.2 °C, range 8.0-8.4 °C) and relative humidity (94.7%, range 93.6-96.6%) of air in this cave were relatively constant. The average PM2.5 and PM10 mass concentration was 5.49 µg m-3 and 11.1 µg m-3. Analysed anions (i.e., F-, Cl-, NO2-, SO42-, PO43- and NO3-) originating largely from the burning of wood and coal for residential heating in nearby villages in the surrounding area. The air in the caves with speleotherapy facilities should be monitored with respect to NTM, PM and anions to ensure a safe environment.

Nontuberculous Mycobacteria Prevalence in Bats' Guano from Caves and Attics of Buildings Studied by Culture and qPCR Examinations.

A total of 281 guano samples were collected from caves (N = 181) in eight European countries (Bulgaria, Czech Republic, France, Hungary, Italy, Romania, Slovakia and Slovenia) and attics in the Czech R. (N = 100). The correlation of detection of mycobacteria between Ziehl-Neelsen (ZN) microscopy and culture examination and qPCR was strong. ZN microscopy was positive in guano from caves (58.6%) more than double than positivity in guano from attics (21.0%; p < 0.01). From 89 mycobacterial isolates (73 isolates from cave guano and 16 isolates from attics' guano), 68 (76.4%) isolates of 19 sp., ssp. and complex were identified as members of three Groups (M. fortuitum, M.chelonae, and M. mucogenicum) and four complexes (M. avium, M. terrae, M.vaccae, and M.smegmatis). A total of 20 isolates (22.5%) belonged to risk group 1 (environmental saprophytes), 48 isolates (53.9%) belonged to risk group 2 (potential pathogens), and none of the isolates belonged to risk group 3 (obligatory pathogens). When comparing bat guano collected from caves and attics, differences (p < 0.01; Mann-Whitney test) were observed for the electrical conductivity, total carbon, total organic, and total inorganic carbon. No difference (p > 0.05; Mann-Whitney test) was found for pH and oxidation-reduction potential parameters.

Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797.

Mycobacterium fortuitum group (MFG) members are able to cause clinical mycobacteriosis in fish and other animals including humans. M. alvei, M. arceuilense, M. brisbanense, M. conceptionense, M. fortuitum, M. peregrinum, M. porcinum, M. senegalense, M. septicum, and M. setense were isolated from fish with mycobacteriosis. In other animals only three MFG species have been isolated: M. arceuilense from camels' milk, M. farcinogenes from cutaneous infections often described as "farcy", and M. fortuitum from different domestic and wild mammals' species. Out of 17, only 3 MFG species (M. arceuilense, M. lutetiense and M. montmartrense) have never been reported in humans. A total of eight MFG members (M. alvei, M. brisbanense, M. conceptionense, M. fortuitum subsp. acetamidolyticum, M. houstonense, M. peregrinum, M. porcinum, and M. septicum) have been isolated from both pulmonary and extrathoracic locations. In extrathoracic tissues five MFG species (M. boenickei, M. farcinogenes, M. neworleansense, M. senegalense, and M. setense) have been diagnosed and only one MFG member (M. fortuitum subsp. acetamidolyticum) has been isolated from pulmonary infection.

Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices.

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9% in Cluster R and 12.6% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

Socio-Economic and Environmental Factors Related to Spatial Differences in Human Non-Tuberculous Mycobacterial Diseases in the Czech Republic.

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that can induce pulmonary and non-pulmonary diseases in susceptible persons. It is reported that the prevalence of NTM diseases is increasing in developed countries, but this differs by regions and countries. NTM species distribution and the rate of diseases caused by NTM vary widely in the historical territories of Moravia and Silesia (Czech Republic). This epidemiologic study of NTM diseases covers the period 2012-2018, reviews isolates obtained from patients with clinical disease and investigates correlations with related socio-economic and environmental factors. Individual NTM patients were included only once during the studied period and results were presented as incidence rate per year. The most frequently isolated NTM meeting the microbiological and clinical criteria in the study were the Mycobacterium avium-intracellulare complex, followed by Mycobacteriumkansasii and Mycobacteriumxenopi. A previously described endemic incidence of M.kansasii in the Karviná district and M.xenopi in the Ostrava district was also observed in this study. The incidence of NTM patients in the whole studied territory was 1.10/100,000 inhabitants (1.33/100,000 in men and 0.88/100,000 in women). The annual incidence of lymphadenitis in children (≤5 years of age) was 2.35/100,000 of the population of children during the 7 year period but increased in the year 2018 to 5.95/100,000. The rate of human tuberculosis in the studied area was 1.97/100,000 inhabitants. The incidence of NTM pulmonary diseases correlated with a lower socio-economic status (r = 0.63) and a higher concentration of benzo[a]pyrene pollution in the air (r = 0.64).

Nontuberculous mycobacteria in the environment of Hranice Abyss, the world's deepest flooded cave (Hranice karst, Czech Republic).

Nontuberculous mycobacteria (NTM) are widely distributed in the environment. On one hand, they are opportunistic pathogens for humans and animals, and on the other hand, they are effective in biodegradation of some persistent pollutants. Following the recently recorded large abundance of NTM in extreme geothermal environments, the aim of the study was to ascertain the occurrence of NTM in the extreme environment of the water zone of the Hranice Abyss (HA). The HA mineral water is acidic, with large concentrations of free CO2, and bacterial slimes creating characteristic mucilaginous formations. Both culture and molecular methods were used to compare the mycobacterial diversity across the linked but distinct ecosystems of HA and the adjacent Zbrasov Aragonite Caves (ZAC) with consideration of their pathogenic relevance. Six slowly growing NTM species (M. arupense, M. avium, M. florentinum, M. gordonae, M. intracellulare) and two rapidly growing NTM species (M. mucogenicum, M. sediminis) were identified in the water and in the dry zones at both sites. Proteobacteria were dominant in all the samples from both the HA and the ZAC. The bacterial microbiomes of the HA mineral water and HA slime were similar, but both differed from the microbiome in the ZAC mineral water. Actinobacteria, a phylum containing mycobacteria, was identified in all the samples at low proportional abundance. The majority of the detected NTM species belong among environmental opportunistic pathogens.

Fluorescence & bioluminescence in the quest for imaging, probing & analysis of mycobacterial infections.

Mycobacterioses represent a global health problem and rapid diagnostic improvements are urgently required. Mycobacteria-specific fluorescence and bioluminescence phenomena have been found to be useful for a wide range of mycobacteria-focused research. Here, we present a critical survey of the most promising techniques in this field and the potential of new methods under investigation. These approaches include acid-fast staining, intrinsic fluorescence of the coenzyme F420, fluorogenic substrates (e.g., ß-lactamase-sensitive coumpounds) and recombination of mycobacteria or mycobacteriophages. Probably the most interesting and emerging host-inspecting approach is in vivo imaging. Detection of fluorescence in vivo, however, is complicated by light scattering, light absorption, and autofluorescence, caused by the tissues. Despite this, many of these systems show promise as the foundations for improved rapid analysis and imaging of mycobacterial infections, both in vitro and in vivo.

MALDI Spectra Database for Rapid Discrimination and Subtyping of Mycobacterium kansasii.

Mycobacterium kansasii is an emerging non-tuberculous mycobacterial (NTM) pathogen capable of causing severe lung disease. Of the seven currently recognized M. kansasii genotypes (I-VII), genotypes I and II are most prevalent and have been associated with human disease, whereas the other five (III-VII) genotypes are predominantly of environmental origin and are believed to be non-pathogenic. Subtyping of M. kansasii serves as a valuable tool to guide clinicians in pursuing diagnosis and to initiate the proper timely treatment. Most of the previous rapid diagnostic tests for mycobacteria employing the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology focused on species-level identification. The purpose of this study was to establish MALDI-TOF MS reference spectra database for discrimination of M. kansasii at the genotype level. A panel of 32 strains, representatives of M. kansasii genotypes I-VI were selected, whole cell proteins extracted and measured with MALDI-TOF MS. A unique main spectra (MSP) library was created using MALDI Biotyper Compass Explorer software. The spectra reproducibility was assessed by computing composite correlation index and MSPs cross-matching. One hundred clinical M. kansasii isolates used for testing of the database resulted in 90% identification at genus-level, 7% identification at species-level and 2% identification was below the threshold of log score value 1.7, of which all were correct at genotype level. One strain could not be identified. On the other hand, 37% of strains were identified at species level, 40% at genus level and 23% was not identified with the manufacturer's database. The MALDI-TOF MS was proven a rapid and robust tool to detect and differentiate between M. kansasii genotypes. It is concluded that MALDI-TOF MS has a potential to be incorporated into the routine diagnostic workflow of M. kansasii and possibly other NTM species.

[Mycobacterium marinum as a cause of human and animal infections].

Mycobacterium marinum is a slowly growing non-tuberculous (environmental, atypical) mycobacterium with zoonotic potential. It occurs in the aquatic environment and causes diseases in fish and other aquatic animals known as mycobacterioses. In humans, it primarily causes skin infections, which are most commonly located in the upper limbs. The disease commonly appears in connection with the aquarium environment and is thus referred to as fish tank granuloma. As with all mycobacterial diseases, treatment is complicated and lengthy. For a definitive determination of the pathogen, biological materials should always be examined in a laboratory specializing in diagnosing mycobacteria. Critical for the right diagnosis is proper sample collection and assessment of the patient's history. To detect mycobacteria, culture and microscopy are generally used. Species are identified using modern biological methods such as mass spectrometry (MALDI), polymerase chain reaction, hybridization probes or sequencing.

Design, Synthesis, and Biological Evaluation of Isothiosemicarbazones with Antimycobacterial Activity.

A series of benzaldehyde and salicylaldehyde-S-benzylisothiosemicarbazones was synthesized and tested against 12 different strains of mycobacteria, Gram-positive and Gram-negative bacteria, and the significant selectivity toward mycobacteria was proved. Twenty-eight derivatives were evaluated for the inhibition of isocitrate lyase, which is a key enzyme of the glyoxylate cycle necessary for latent tuberculosis infection, and their iron-chelating properties were investigated. Two derivatives, 5-bromosalicylaldehyde-S-(4-fluorobenzyl)-isothiosemicarbazone and salicylaldehyde-S-(4-bromobenzyl)-isothiosemicarbazone, influenced the isocitrate lyase activity and caused a better inhibition at 10 µmol/L than 3-nitropropionic acid, a standard inhibitor. The compounds were also found to act as exogenous chelators of iron, which is an obligate cofactor for many mycobacterial enzymes. Due to their low cytotoxicity, together with the activity against isocitrate lyase and the ability to sequester iron ions, the compounds belong to potential antibiotics with the main effect on mycobacteria.

Avian Mycobacteriosis: Still Existing Threat to Humans.

The nontuberculous mycobacteria are typically environmental organisms residing in soil and water. These microorganisms can cause a wide range of clinical diseases; pulmonary disease is most frequent, followed by lymphadenitis in children, skin and soft tissue disease, and rare extra pulmonary or disseminated infections. Mycobacterium avium complex is the second most common cause of pulmonary mycobacterioses after M. tuberculosis. This review covers the clinical and laboratory diagnosis of infection caused by the members of this complex and particularities for the treatment of different disease types and patient populations.

Mycobacteria in water used for personal hygiene in heavy industry and collieries: a potential risk for employees.

Environmental mycobacteria (EM) constitute a health risk, particularly for immunocompromised people. Workers in heavy industry and in collieries represent an at-risk group of people as their immunity is often weakened by long-term employment in dusty environments, frequent smoking and an increased occurrence of pulmonary diseases. This study was concerned with the presence of EM in non-drinking water used for the hygiene of employees in six large industrial companies and collieries. Over a period of ten years, 1096 samples of surface water treated for hygiene purposes (treated surface water) and treated surface water diluted with mining water were examined. EM were detected in 63.4 and 41.5% samples of treated surface water and treated surface water diluted with mining water, respectively. Mycobacterium gordonae, M. avium-intracellulare and M. kansasii were the most frequently detected species. Adoption of suitable precautions should be enforced to reduce the incidence of mycobacteria in shower water and to decrease the infectious pressure on employees belonging to an at-risk group of people.

Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase.

The global burden of tuberculosis, its health and socio-economic impacts, the presence of drug-resistant forms and a potential threat of latent tuberculosis should serve as a strong impetus for the development of novel antituberculosis agents. We reported the in vitro activity of salicylanilide benzoates and pyrazine-2-carboxylates against Mycobacterium tuberculosis (minimum inhibitory concentrations as low as 0.5 µmol/L). Nineteen salicylanilide derivatives with mostly good antimycobacterial activity were evaluated for the inhibition of two essential mycobacterial enzymes, methionine aminopeptidase and isocitrate lyase, which are necessary for the maintenance of the latent tuberculosis infection. Salicylanilide derivatives act as moderate inhibitors of both mycobacterial and human methionine aminopeptidase and they also affect the function of mycobacterial isocitrate lyase. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate was the most potent inhibitor of mycobacterial methionine aminopeptidase (41% inhibition at 10 µmol/L) and exhibited the highest selectivity. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide and 4-chloro-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate caused 59% inhibition of isocitrate lyase at 100 µmol/L concentration and (S)-4-bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 2-acetamido-3-phenylpropanoate produced 22% inhibition at 10 µmol/L; this rate is approximately comparable to 3-nitropropionic acid. Inhibition of those enzymes contributes at least in part to the antimicrobial activity of the compounds.

Tetracycline resistance and presence of tetracycline resistance determinants tet(V) and tap in rapidly growing mycobacteria from agricultural soils and clinical isolates.

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates' BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research.