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Non-tuberculous mycobacteria (NTM), also known as an environmental and atypical mycobacteria, can cause the chronic pulmonary infectious diseases. Macrophages have been suggested as the main host cell to initiate the innate immune responses to NTM infection. However, the molecular mechanism to regulate the antimicrobial immune responses to NTM is still largely unknown. Current study showed that the NTM clinical groups, Mycobacterium abscessus and Mycobacterium smegmatis, significantly induced the M1 macrophage polarization with the characteristic production of nitric oxide (NO) and marker gene expression of iNOS, IFNγ, TNF-α, IL1-β and IL-6. Interestingly, a non-histone nuclear protein, HMGN2 (high-mobility group N2), was found to be spontaneously induced during NTM-activated M1 macrophage polarization. Functional studies revealed that HMGN2 deficiency in NTM-infected macrophage promotes the expression of M1 markers and the production of NO via the enhanced activation of NF-κB and MAPK signalling. Further studies exhibited that HMGN2 knock-down also enhanced IFNγ-induced M1 macrophage polarization. Finally, we observed that silencing HMGN2 affected the survival of NTM in macrophage, which might largely relevant to enhanced macrophage polarization into M1 phenotype under the NTM infection. Collectively, current studies thus suggested a novel function of HMGN2 in regulating the anti-non-tuberculous mycobacteria innate immunity of macrophage., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.

Background: Nontuberculous mycobacteria (NTM) are common bacteria in water and especially water supply distribution systems. Some species can cause infections, especially in immunocompromised patients and other risk groups. This study examined the frequency of occurrence of NTM in 135 household potable water samples collected from household water taps in Tenerife Island., Methods: Mycobacteria species were identified by polymerase chain reaction targeting the 16S rRNA and 16S-23S rRNA regions, and by double-reverse hybridization on a dipstick using colloidal gold-bound and membrane-bound probes (Speed-Oligo(®) Mycobacteria). Some species were identified by sequencing the gene that encodes the 16S rRNA region., Results: NTM were present in 47.4% of the samples. Mycobacterium fortuitum was the NTM isolated most frequently (70.3%), followed by Mycobacterium canariasense (6.3%) and Mycobacterium chelonae (6.3%). Other species were isolated at lower percentage frequencies., Conclusion: We isolated and identified the species M. canariasense in water supplies for public consumption. This species has previously been reported only in hospital settings. The elevated presence of NTM in the water supply indicates that it may be a reservoir for infections caused by recently described species of mycobacteria., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Disease caused by nontuberculous mycobacteria (NTM) is increasing in frequency. The outcome of treatment for NTM lung disease is poor, particularly lung disease caused by Mycobacterium simiae and M. abscessus. Exploring synergy between active available drugs is a sensible way forward given the lack of new active drugs. We tested for synergy between amikacin and clofazimine, using standardized methods, in 564 consecutive clinical isolates identified as 21 species of rapidly growing mycobacteria, 16 clinical M. avium complex isolates, and 10 M. simiae isolates. Clofazimine and amikacin are each active in vitro against NTM; 97% (n = 548) of the rapid growers revealed MICs of clofazimine of ≤1 μg/ml, and 93% (n = 524) proved susceptible to amikacin. The combination showed significant synergistic activity in 56 of 68 (82%) eligible M. abscessus isolates, 4 of 5 M. chelonae isolates, and 1 M. fortuitum and 1 M. cosmeticum isolate, with 4- to 8-fold decreases in MICs to both drugs. Significant synergy could also be demonstrated against all M. avium complex and M. simiae isolates, with fractional inhibitory concentrations of <0.5. Clofazimine and amikacin show significant synergistic activity against both rapidly and slowly growing nontuberculous mycobacteria. The safety and tolerability of adding clofazimine to amikacin-containing regimens should be tested in clinical trials, and the results of susceptibility tests for these two compounds and their combination merit clinical validation. Synergy between clofazimine and other antibiotics with intracellular targets should be explored.

Rapidly growing mycobacteria (RGM) are ubiquitous in nature and widely distributed in water, soil and animals. During the past three decades we have observed a notable increment of infections caused by RGM, both localized and disseminated, as well as nosocomial outbreaks of contaminated medical equipment. The microbiological diagnosis of RGM infections includes direct microscopic observation and culture. The taxonomic identification is performed by phenotypic, biochemical, chromatographic and molecular biology techniques. The treatment differs from that of other mycobacteriosis like tuberculosis, owing to the variable in vitro susceptibility of the species of this group. The RGM are resistant to conventional antituberculous drugs, but can be susceptible to broad spectrum antimicrobial agents. In this study we comment on the significant aspects of human infections by RGM, including their biology, epidemiology, pathology, microbiological diagnosis, taxonomic identification, antimicrobial susceptibility and treatment., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

The reliability of the BacT/Alert 3D unit for automated detection of nontuberculous mycobacteria (NTM) that grow optimally at 30 °C was assessed. This system reliably maintained a temperature of 30 °C and detected 50% of the clinical NTM strains (5 Mycobacterium marinum and 3 Mycobacterium gordonae strains) faster than 37 °C culture.

The MB/BacT system (MB/BacT) with a revised antibiotic supplement kit was compared with the BACTEC 460 system (BACTEC 460) in a test of 488 specimens submitted for mycobacterial culture from 302 patients. Twenty-four Mycobacterium tuberculosis isolates were detected by the BACTEC 460 versus 23 isolates by the MB/BacT. Mean time until detection of M. tuberculosis isolates identified by both systems was 11.9 days for the BACTEC 460 versus 13.7 days for the MB/BacT (P = 0.046). M. avium complex was detected in 12 specimens by the MB/BacT versus 10 specimens by the BACTEC 460. Only 8 of 14 (57%) M. avium isolates were detected by both systems, with a mean time until detection of 10.1 days for the BACTEC 460 and 14.2 days for the MB/BacT (P = 0.009). The BACTEC 460 and the MB/BacT detected M. gordonae in four specimens, but only a single specimen was positive by both systems. One M. fortuitum isolate and one of five M. kansasii isolates were recovered only by the BACTEC 460. The bacterial overgrowth rate was 7.0% for the MB/BacT versus 4.1% for the BACTEC 460. We found the MB/BacT to be comparable to the BACTEC 460 for mycobacterial detection. Even though time until detection with the MB/BacT was slightly longer (1.8 days longer for M. tuberculosis and 4.1 days for M. avium [mean values]) and the bacterial overgrowth rate was somewhat higher, the decreased labor, the availability of a computerized data management system, and the noninvasive, nonradiometric aspects of the MB/BacT offset these relative disadvantages and make it an acceptable alternative for use in the diagnostic laboratory.

Detection and identification of mycobacterial species using DNA-rRNA probes from colonies has been used for some time. Guidelines for probe use with a specific minimum growth index (GI) cutoff from BACTEC 12B bottles or its use with 7H9 broth has not been defined. This study defined this minimum GI guideline. Clinical specimens received during the study period were decontaminated and directly inoculated into BACTEC 12B bottles and appropriate solid media. An initial probe test was performed at a GI reading of > or = 100. An additional probe test was made at GI of > or = 300. A total of 1377 specimens were processed with 98 specimens containing 99 isolates of Mycobacterium. Of these, 82 were M. avium complex (MAC), six were M. tuberculosis (Mtb), and 11 were other species. At a GI of > or = 100, 82% of MAC were detected; at a GI of > or = 300, 89% were detected. All Mtb were detected at a GI of > or = 100. The probes correctly identified all isolates from backup 7H9 broths. There were no false-positive probe results for any of the isolates from either BACTEC 12B or 7H9 media. Therefore, the DNA-rRNA probe test at a GI of > or = 300 saves considerable time (on average, 21 days saved) for the detection and identification of Mtb and MAC. This time saving compares with waiting for the production of colonies while retaining acceptable sensitivity and excellent specificity.

Exogenous lipid pneumonia following bronchography occurred in a woman with allergic bronchopulmonary aspergillosis (ABPA) whose bronchi had been colonized with Mycobacterium chelonei chelonei for the previous year. The lung biopsy specimen disclosed organisms within lipid vacuoles in the areas of lipid pneumonia, while caseating granulomas, the usual tissue reaction to pathogenic mycobacteria, were absent. These pathologic findings suggested that the infection with M chelonei was noninvasive, and we were able to continue the corticosteroid therapy that was necessary for control of the underlying ABPA.

With the increasing significance of group IV atypical mycobacteria as etiological agents in a variety of infections, studies were conducted to determine their growth capabilities in water and their comparative resistance to disinfectants used to decontaminate hospital equipment. Isolates of Mycobaterium chelonei (TM strains) from peritoneal fluids of patients and peritoneal dialysis machines were able to multiply in commercial distilled water, with generation times at 25 degrees C ranging from 8 to 15 h. Levels of 10(5) to 10(6) cells per ml were attained, and these stationary-phase populations declined only slightly over a 1-year period. Results of studies to determine resistance to disinfectants showed the following. (i) TM strains of M. chelonei cultured in commercial distilled water showed survivors in 2% aqueous formaldehyde (HCHO) solutions up to 24 h; in 8% HCHO, only a 2-log reduction in viable counts was observed over a 2-h sampling period. Reference ATCC strains of M. chelonei and M. fortuitum were rapidly inactivated, with no survivors after 2 h of exposure to 2% HCHO or 15 min of exposure to 8% HCHO. (ii) In 2% alkaline glutaraldehyde, TM strains survived 60 min. whereas ATCC strains showed no survivors after 2 min of contact time. (iii) All M. chelonei and M. fortuitum strains survived 60 min of exposure to concentrations of 0.3 and 0.7 microgram of free chlorine per ml at pH 7.

Cultural and biological tests were carried out on the livers and spleens of three wood pigeons. Acid-fast organisms were observed in histological sections and smears taken from tuberculosis-like lesions in the organs. Biological tests revealed that all the isolates sensitised the guinea pig to avian tuberculin. One was pathogenic for chickens while the remaining two were non-pathogenic. All were non-pathogenic for rabbits and domestic pigeons. The organisms could not be cultured on conventional media for Mycobacterium tuberculosis or media containing mycobactin. However they were readily maintained on 7H11 media. These results indicate a separate species of mycobacterium related to M avium.

Re-evaluation of a previous study dealing with atypical mycobacteria grown from specimens of bone marrow provided strong evidence that the isolates were of no clinical significance. Bone marrow culture should not be undertaken as a diagnostic test for pulmonary atypical mycobacteriosis.

Infections having atypical mycobacteria are occurring more frequently for 2 main reasons. First there are the disturbances of the immunity system, whether due to the acquired immune deficiency syndrome (AIDS) or the result of therapeutic measures taken against malignant processes, other diseases or after organ transplantation. Second, travel and free-time activities increase the risk of an infection. In many cases such infections become localized on the skin, but can also develop changes in the lungs, depending on the patient's occupation. For this reason we provide a review of the new literature on the microbiology of mycobacteria as well as on the symptoms of diseases of the skin and other organs which are caused by atypical mycobacteria, such as swimming pool granuloma or Buruli ulcer.

Growth of Mycobacterium kansasii TMC 1203 in B6D2 F1 hybrid mice was associated with increased splenic cellular proliferation, hyperplasia and the generation of non-specific antibacterial resistance. Both responses were dose dependent; the larger the inoculum, the more rapid and extensive the cellular response. However, such mice were still unable to reduce the mycobacterial load within the tissues, apparently because of their inherent resistance to inactivation by immunologically activated macrophages. On the other hand, mice infected with the non-persistent strain of M. kansasii 1214 exhibited only a transient increase in non-specific (anti-listeria) resistance which rapidly declined as the number of viable mycobacteria within the spleen fell below an arbitrary threshold level. Mice infected with either M. kansasii 1203 or 1214 could be immunized with sheep red blood cells (SRBCs), an unrelated T cell-dependent antigen. The humoral (PFC) response was not affected by the mycobacterial load within the spleen. However, the delayed footpad swelling reaction was severely depressed. The latter could be restored merely by increasing the size of the intravenous sensitizing inoculum 100-fold. The present study indicates that mice chronically infected with M. kansasii are not severely immunosuppressed (as had been inferred from earlier in vitro lymphoproliferation studies) but are fully capable of responding to appropriate in vivo stimuli.

Eighty-nine isolates of rapidly growing mycobacteria associated with cardiac bypass-related infections were characterized. Isolates from sporadic infections belonged to eight taxonomic groups and displayed numerous multilocus enzyme genotypes, plasmid profiles, and heavy metal and antibiotic resistance patterns. Compared with 449 noncardiac wound isolates, 45 sporadic cardiac isolates were more likely to be Mycobacterium fortuitum and M. smegmatis and less likely to be M. chelonae. About 80% of cardiac and noncardiac isolates were from southern coastal states. Eight outbreaks of cardiac bypass-related infections were identified. Strains from each outbreak were genotypically distinctive, and five outbreaks involved more than one strain. In two outbreaks, isolates from environmental sources and noncardiac infections were similar or identical to isolates from sternal wound infections. The heterogeneity of these isolates suggests that most isolates are unrelated and are derived from local environmental sources rather than from contaminated commercial surgical materials or devices.

Germfree nude mice (NMRJ-nu/nu) and germfree nu/+ mice were infected with Mycobacterium marinum in the footpad test. After pretreatment with 400 mg/kg cyclophosphamide, significantly higher amounts of M. marinum were reached in the footpad and in the liver. On the other hand, the yield of M. marinum could not be elevated by in vivo inhibition of macrophages with silica.

We report here the first laboratory studies simulating the conditions for natural aerosolization of Mycobacterium intracellulare and M. scrofulaceum and estimate the yields for this pathway of transfer of pathogenic mycobacteria from water to air; M. intracellulare and M. scrofulaceum were both concentrated in droplets ejected from cell suspensions of densities comparable to those found in natural freshwaters (100 to 2,000 colony-forming units per ml). The enrichment factor (defined as the concentration of cells per droplet volume divided by the concentration of cells in the bulk suspension per equivalent volume) for M. intracellulare isolates ranged from 68 to 15,000, with an average of 2,922; for M. scrofulaceum it ranged from 35 to 550, with an average of 177. One factor responsible for the greater aerosolization of M. intracellulare was their aggregation. However, after vortexing, M. intracellulare were still aerosolized more (enrichment factor, 325) than M. scrofulaceum. Increasing salt concentrations enriched the aerosolization of both species, but the number of organisms transferred from water to air did not increase proportionately because the salt decreased the droplet volume. Other waterborne pathogens such as Legionella pneumophila were also enriched and transferred from water to air, indicating that this pathway for possible infection of humans may also be significant for other respiratory diseases.

In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation.

Rapidly growing mycobacteria of two species, i.e. M. fortuitum and M. chelonei caused in humans mycobacteriosis frequently similar in its manifestations to tuberculosis. The both mycobacterial species were resistant to the majority of antituberculous drugs and sensitive to amikacin, dioxycycline, sefmetazole and some sulfanilamides. It was shown that it was possible to identify the mycobacterial species with the routine tests and paper indicator systems.

Thirteen cases of disseminated Mycobacterium avium-intracellulare (MAI), representing a total of 27 bone marrow specimens, were studied. The patients ranged from 25 to 46 years of age and included ten immunocompromised and three immunocompetent hosts. Peripheral blood findings included anemia in all patients, leukopenia in 73%, thrombocytopenia in 45%, and pancytopenia in 45%. Fourteen of the specimens (52%) showed granulomas ranging from small, subtle lymphohistiocytic aggregates to larger lymphohistiocytic lesions and clusters of epithelioid histiocytes; almost half of these lesions were missed initially. Rare acid-fast bacilli were seen in only one case, but 53% grew MAI on culture. In one case of the acquired immunodeficiency syndrome, culture was positive in the absence of inflammation or AFB on staining. These findings are not significantly different from those reported in disseminated Mycobacterium tuberculosis infection.

A comparison of clinical and environmental (e.g., soil, dust, water, and aerosol) isolates of the Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) group has been made. The frequency of M. avium-intracellulare clinical isolates able to grow without OADC enrichment, able to grow at 43 degrees C, or express resistance to streptomycin or cadmium was significantly higher than those among environmental isolates. Only the frequency of clinical M. scrofulaceum isolates able to grow at 43 degrees C and without OADC enrichment was significantly higher than that of environmental isolates. Because of the high frequency of clinical M. avium-intracellulare isolates able to grow without OADC, grow at 43 degrees C, and resistant to streptomycin, these 3 characteristics are suggested for use as epidemiologic markers for MAIS. There were no significant differences between clinical M. avium-intracellulare isolates from 3 widely separated geographic regions. Among M. avium-intracellulare environmental isolates, only those collected in droplets above bodies of water (i.e., aerosols) shared those characteristics unique to clinical MAIS. That observation suggests that these aerosols are a likely source of human MAIS infection. The ability of isolates to grow at 43 degrees C and without OADC enrichment was highly correlated. Gentamicin-, streptomycin-, and D-cycloserine-resistances were also frequently found together. Isolates carrying plasmids and either able to grow at 43 degrees C, without OADC enrichment, or mercury-resistant were found more frequently than expected as well. This suggests their genetic determinants are plasmid-encoded.

Mycobacterium simiae has been isolated from a patient with a pulmonary infection. During 1 1/2 years 10 different isolates of this strain could be cultivated. In this case it has not been proved that Mycobacterium simiae was the etiological agent, although the probability is very high.

Although techniques based on immunologic or chromatographic analyses have been described for identifying mycobacteria in clinical laboratories, most microbiologists continue to rely on a series of specialized physiological and biochemical tests for this purpose. The recognition of additional significant species over the past decade has required the addition of more tests to the battery used for mycobacterial identification. This paper will review briefly the taxonomic status of species likely to be encountered in clinical specimens and the most useful tests for characterizing them. Strategies will be presented for using these tests in the most efficient way to provide optimal resolution of taxa without use of an unreasonably large battery of tests. A brief survey of techniques that may become more practical in the future will also be included.

In summary, rapidly growing mycobacteria, M. fortuitum and M. chelonei, are pathogens of increasing importance which are often hospital-acquired and can infect patients with iatrogenic immunosuppression. They readily grow on routine mycobacterial culture media and must be distinguished from non-pathogenic rapidly growing species and slowly growing mycobacteria. Widely distributed in nature, they are often present in hospital environments, especially in water. Compared to M. tuberculosis they are weak pathogens, and infected patients are not considered contagious. Disease is probably acquired from environmental sources by direct entry of the organisms through traumatized skin or mucous membranes or by aspiration into previously abnormal lungs. They are usually resistant to antituberculous agents but are susceptible to several commonly used antibacterial agents. Treatment generally requires one or more active antibiotics plus adjunctive surgery in many cases. Prevention of nosocomial infection lies in proper disinfection of potentially contaminated medical devices and elimination of contaminated water.

Patients with acquired immune deficiency syndrome may develop infection with mycobacteria, particularly Mycobacterium avium-M. intracellulare (MAI). These infections can frequently be associated with demonstrable mycobacteremia with the organism. In this study, we compared the sensitivity of a radiometric (BACTEC system; Johnston Laboratories, Inc., Towson, Md.) liquid medium culture system with that of conventional solid mycobacterial culture media for cultures of blood from these patients. Both systems were inoculated with blood concentrate prepared by lysis-centrifugation (Isolator; Du Pont Co., Wilmington, Del.). Of 46 acquired immune deficiency syndrome patients whose blood was cultured, 28% had cultures positive for MAI. Patients had from less than 1 to more than 100 MAI colonies per ml of blood. Lowenstein-Jensen and Middlebrook 7H11 agars were comparable in recovery of MAI. BACTEC 12A vials containing double the standard volume of medium (4 ml) were more sensitive and were positive slightly earlier than vials containing the standard volume (2 ml). Conventional media detected 98% of positive cultures; BACTEC vials containing double volumes of medium detected 94% of positive cultures, whereas single-volume vials detected 77%. BACTEC vials were positive approximately 5 to 6 days sooner than slants or plates containing conventional media. For a few cultures, the use of unconcentrated blood was compared with the use of Isolator-concentrated blood by using each of these as inocula for BACTEC vials. Results for these cultures suggested that, although the use of Isolator-concentrated blood resulted in greater sensitivity than the use of unconcentrated blood would, the use of unconcentrated blood would still result in the detection of at least 78% of positive cultures.

Five cases of infection due to an unusual mycobacterium are recorded, and the growth requirements and histological findings are discussed. On initial culture, these mycobacteria could be grown only on Lowenstein-Jensen medium containing ferric ammonium citrate, and the histology of the skin lesions was not always typical of mycobacterial disease.