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PubMed2026

Development of an arabinose-inducible gene expression system for nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.

Arabinose↗

PubMed2003

Nontuberculous mycobacteria in hospital water systems: application of HPLC for identification of environmental mycobacteria.

Nontuberculous mycobacteria (NTM), ubiquitous in water environments, are increasingly recognized as nosocomial pathogens. Our study reports a one-year survey of the water system of two hospitals, A and B, in a small town near Florence, Italy. NTM were found throughout the study period in both settings, but B showed a significantly higher mycobacterial load. Mycobacterium gordonae and Mycobacterium fortuitum were the most frequent species isolated. Identification was carried out by conventional techniques and by high performance liquid chromatography (HPLC) analysis of cell wall mycolic acids. HPLC profiling could be used as a first-choice method for identification of environmental mycobacteria.

Chromatography, High Pressure Liquid↗

PubMed2005

[Laboratory diagnosis and antibiotic susceptibilities of nontuberculous mycobacteria].

Nontuberculous mycobacteria (NTM) are found widespread in nature. They can live in soil, water supplies, plants and various environmental conditions. Today there are nearly 100 known NTM species and more than 50 of them are associated with human diseases. NTM can cause pulmonary diseases, lymphadenitis, skin, soft tissue and skeletal infections, catheter related bloodstream infections and disseminated infections in patients with underlying diseases like AIDS, chronic obstructive pulmonary diseases, emphysema, bronchiectasis, cystic fibrosis and chronic alcoholism. The laboratory diagnosis of NTM has improved in parallel to the improvements in molecular biology. The new DNA sequencing and microarray technologies have been started to use in laboratories. Antibiotic susceptibility tests can be used both for taxonomic and clinical purposes. In this review article, the importance of laboratory diagnosis and antibiotic susceptibilities of NTM have been discussed.

Environmental Microbiology↗

PubMed2001

Nosocomial infections due to nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and cause colonization, infection, and pseudo-outbreaks in health care settings. Data suggest that the frequency of nosocomial outbreaks due to NTM may be increasing, and reduced hot water temperatures may be partly responsible for this phenomenon. Attention to adequate high-level disinfection of medical devices and the use of sterile reagents and biologicals will prevent most outbreaks. Because NTM cannot be eliminated from the hospital environment, and because they present an ongoing potential for infection, NTM should be considered in all cases of nosocomial infection, and careful surveillance must be used to identify potential outbreaks. Analysis of the species of NTM and the specimen source may assist in determining the significance of a cluster of isolates. Once an outbreak or pseudo-outbreak is suspected, molecular techniques should be applied promptly to determine the source and identify appropriate control measures.

Cross Infection↗

PubMed1984

Nontuberculous mycobacteria.

Nontuberculous mycobacteria are identified in almost half of the cultures reported positive for mycobacteria in clinical laboratories in the United States. While many represent saprophytic colonization or laboratory contamination, a significant number of these organisms are the agents of disease. Such organisms can be the cause of pulmonary, soft tissue, cutaneous and lymphatic infections, keratitis, osteomyelitis, postsurgical infection, endocarditis and disseminated disease.

Humans↗

PubMed1988

Pulmonary infection with nontuberculous mycobacteria.

Nontuberculous mycobacterial infections (NTM) are being increasingly recognized as a cause of chronic pulmonary disease. We recently reviewed the clinical, radiologic, and bacteriologic presentation of 89 adult patients ill enough to have been hospitalized between 1981 and 1985 with the diagnosis of NTM. Preexisting lung disease was present in 82% and alcohol abuse in 40%. Although M. avium complex was identified in 51% of the patients, M. xenopi, which is usually reported to occur infrequently, accounted for 38% of our cases and M. kansasii for only 9%. Treatment was limited by a high incidence of associated disease, in vitro drug resistance, drug toxicity, and a mortality rate of 32% within 18 months of admission. Nevertheless, bacteriologic conversion occurred in 29% of those treated. M. xenopi appears to be an important pathogen in southern Ontario. It differs from the other NTM by having a different pattern of in vitro drug resistance but not by its clinical or radiologic presentation.

Adult↗

PubMed1990

Pulmonary disease caused by nontuberculous mycobacteria.

Nontuberculous mycobacterial pulmonary infections have become more common in recent years. The diagnosis is often overlooked because the findings may be subtle or because the radiographic appearance may change slowly or not at all for long periods of time. As a rule, the radiographic findings of nontuberculous mycobacterial pulmonary infections are identical to those of tuberculosis in any given patient. Cavitary disease in nontuberculous mycobacterial infections is less common than in tuberculosis. The most common radiographic finding is one or more areas of clustered fibroproductive nodules that change slowly. Mycobacterium kansasii infection responds well to therapy, whereas M avium-intracellulare infection is difficult to treat. Awareness of the radiographic appearance of the nontuberculous mycobacterial pulmonary infections will facilitate their diagnosis so that appropriate therapy may be initiated before the disease is far advanced.

Diagnosis, Differential↗

PubMed1994

[The isolation of nontuberculous mycobacteria and the pulmonary infections of nontuberculous mycobacteria in the general hospital].

In the past eight years, we experienced 94 cases of nontuberculous mycobacteria (NTM) isolation from the specimens of sputum, gastric juice, pleural fluid and fiberoptic bronchoscopic procedure at Chiba Kaihin Municipal Hospital. The species of NTM were M. avium complex (MAC) in 23, M. gordonae in 15, M. kansasii in 12, M. fortuitum in 8, M. chelonae in 7, etc.. The number of isolation of NTM has increased, and it is worthwhile to mention that it had exceeded the number of isolation of M. tuberculosis since 1991. We experienced 28 cases of pulmonary infections caused by NTM during the same period. Thirteen cases were by M. avium complex, 11 cases by M. kansasii, one case by MAC and M. kansasii, one case by M. chelonae and two cases by unidentified NTM. Pulmonary infections by NTM also gradually increased. Thirteen cases (46.4%) had underlying diseases in the respiratory system. Most of them were progressive diseases with cavities and were easily diagnosed by mycobacterial examinations of the specimens such as sputum or gastric juice. We were unable to diagnose 12 cases (42.9%) by mycobacterial examinations. Most of them had no underlying diseases and showed small lesions without cavitation on the chest X-ray. Eight cases were diagnosed by the histopathological examination of TBLB (transbronchial lung biopsy) specimens. In such cases, these procedures were also useful for the early diagnosis. This study indicated the increase of the isolation of NTM and the pulmonary infections by NTM. Once the infections are established, they are progressive, and the early diagnosis of these infections are thought to be important.

Adult↗

PubMed2001

Nontuberculous mycobacteria.

The nontuberculous mycobacteria are for the most part ubiquitous environmental organisms that only rarely cause disease in humans. Therefore, the normal host defense against these organisms must be quite robust, as exposure is universal and disease is rare. The organisms that are most commonly encountered in clinical practice, Mycobacterium avium, M. intracellulare, M. kansasii, M. fortuitum, M. abscessus, and M. chelonae, are frequently found in water sources and soil. These organisms share significant structural and biochemical similarities with their more pathogenic relative, M. tuberculosis (MTB). Because they are of significantly lower pathogenicity than MTB, patients with abnormal susceptibility to these infections should include those with defects that may be identifiable. Study of these patients should lead to determination of the mechanisms underlying resistance to these organisms, which in turn are likely to be highly informative regarding host defense against these infections and their more virulent relative MTB. Furthermore, recognition of host factors that permit infection with nontuberculous mycobacteria in otherwise normal hosts will identify pathways that can be targeted for therapeutic intervention. Thus, the search for genetic and acquired susceptibility to nontuberculous mycobacteria is also a search for susceptibility factors for MTB as well as an opportunity to recognize endogenous pathways that can be exploited therapeutically.

Anti-Bacterial Agents↗

PubMed1997

Proficiency testing of clinical microbiology laboratories using modified decontamination procedures for detection of nontuberculous mycobacteria in sputum samples from cystic fibrosis patients. The Nontuberculous Mycobacteria in Cystic Fibrosis Study Group.

An improved decontamination method has been demonstrated to reduce overgrowth of mycobacterial media by Pseudomonas aeruginosa and allow the successful recovery of nontuberculous mycobacteria (NTM) from cystic fibrosis (CF) patients. Twenty microbiology laboratories participating in a multicenter investigation designed to determine the significance of NTM in CF patients were required to demonstrate proficiency in the incorporation of this improved method; this was accomplished by successful decontamination and culture workup of a panel of simulated sputum samples seeded with P. aeruginosa and various NTM. All laboratories successfully recovered NTM from samples with acid-fast bacillus (AFB) smear scores of 3+/4+ (i.e., 2 to 18 or > 18 organisms/field). Low-inoculum samples (1+/2+ AFB smears [2 to 18 organisms in 100 or 10 fields]) were problematic in that processed specimens were often smear and/or culture negative.

Bacteriological Techniques↗

PubMed1995

The changing spectrum of pulmonary infections due to nontuberculous mycobacteria.

Although nontuberculous mycobacteria (NTM) infections are not common, there is evidence that they are becoming increasingly recognized and in some parts of the country occur more frequently than M. tuberculosis. The classic description of NTM pulmonary infection is that of a male patient with cavitary infiltrates and predisposing lung disease. With an increase in the number of reported cases of infection, there have been other syndromes described, particularly in elderly female patients without any predisposing conditions, those with asymptomatic pulmonary nodules, esophageal disease causing recurrent aspiration pneumonitis, and disseminated infection in AIDS. Therapy of M. avium complex (MAC) disease is far from definitive whereas infections due to M. kansasii can be effectively treated even in the presence of profound immunosuppression. Recognition of these infections and their recent change in epidemiology hopefully will encourage practitioners to enroll their patients into clinical protocols that will help to establish the optimal regimen(s).

Causality↗

PubMed1997

Nontuberculous mycobacteria.

The nontuberculous mycobacteria (NTM), especially Mycobacterium avium complex, are being recognized with increasing frequency as clinical pathogens, not only as a cause of disseminated disease in patients with AIDS but also as a cause of chronic lung disease in patients without AIDS. These infections have traditionally been difficult and frustrating to treat; however, the introduction of new agents, such as clarithromycin, azithromycin, and rifabutin, has significantly improved outcome for patients with some NTM infections. The new therapeutic regimens can be associated with severe toxicities and drug interactions that dictate the need for careful monitoring of patients undergoing treatment for M. avium complex disease. Clinicians will be called on with increasing frequency to determine the significance of NTM isolated from their patients. This determination will require knowledge about the pathogenicity and the appropriate therapy of a variety of NTM species.

AIDS-Related Opportunistic Infections↗

PubMed2005

Species identification of mycobacteria in paraffin-embedded tissues: frequent detection of nontuberculous mycobacteria.

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium-intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.

Base Sequence↗

PubMed1980

Epidemiology of infection by nontuberculous Mycobacteria. III. Isolation of potentially pathogenic mycobacteria from aerosols.

Nontuberculous mycobacteria (21 isolates), biochemically similar to those that are recovered from humans, were recovered from rainwater and from natural river waters and their aerosols in the area of Richmond, Virginia. Field experiments have confirmed the existence of a natural mechanism for the transfer of significant numbers of mycobacteria from water to air. These findings support the hypothesis that aerosolization of potentially pathogenic mycobacteria from waters of the southeastern United States may be a major pathway for human infection.

Aerosols↗

PubMed1996

Epidemiology of disease caused by nontuberculous mycobacteria.

The clinically important nontuberculous mycobacteria include M. kansasii, M. genavense, M. marinum, M. simiae, M. scrofulaceum and M. szulgai, M. avium, M. haemophilum, M. intracellulare, M. malmoense, M. ulcerans, and M. xenopi, M. abscessus, M. chelonae, M. fortuitum, and (rarely) M. smegmatis. Four clinical syndromes account for nearly all cases: pulmonary disease, lymphadenitis, skin or soft tissue disease, and disseminated disease in AIDS. M. avium and M. intracellulare (known together as M. avium complex), are the most common causes of pulmonary disease, lymphadenitis, and disseminated disease. All four clinical syndromes seem to be increasing in frequency, particularly in immunosuppressed hosts. Nontuberculous mycobacteria are acquired from the environment, but specific reservoirs of these organisms leading to human disease have not been defined.

Humans↗

PubMed2003

Molecular diagnosis of nontuberculous mycobacteria.

PURPOSE OF REVIEW: Diagnosis of infection due to nontuberculous mycobacteria is not easy, as it must be distinguished from colonization or contamination by other nontuberculous mycobacteria. Molecular methods offer many advantages over conventional methods of identification. The results are obtained rapidly, are reliable and reproducible, and even mixed or contaminated cultures can be examined. This review highlights the recent advances in molecular techniques for identification of nontuberculous mycobacteria. RECENT FINDINGS: Nontuberculous mycobacteria are ubiquitous towards the environment and have the potential to colonize and cause serious infection. An increasing number of species and clinical presentations are being described, and progress has been made towards the understanding of the underlying predisposing factors. Disease caused by nontuberculous mycobacteria is often associated with various forms of immunosuppression, particularly HIV infection, whereas mild forms of immune defects have been observed in some patients who, apart from their nontuberculous mycobacterial disease, seem to be healthy on initial examination. Molecular techniques have shown their usefulness for the identification of most mycobacteria. Probes are widely used in clinical laboratories for the identification of the most common mycobacterial species. Because automated DNA sequencing and the programs for analysing sequence data have become technically simpler, polymerase chain reaction-based sequencing is now used in many mycobacterial reference laboratories as a routine method for species identification. SUMMARY: Significant advances have been made with molecular tools for diagnosis of mycobacteria. The DNA microarray technique holds great promise for the future because it is easy to perform, it can be readily automated, and it allows the identification of a large number of mycobacterial species in one reaction.

Bacterial Typing Techniques↗

PubMed2002

[Multicenter evaluation of a newly developed microdilution test, brothMIC NTM to determine minimum inhibitory concentrations of antimicrobial agents for nontuberculous mycobacteria].

We developed a new broth microdilution susceptibility test for nontuberculous mycobacteria to determine minimum inhibitory concentrations(MICs). The test method utilized air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The nine agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, clarithromycin, ethionamide and amikacin. The test plates were reconstituted by inoculation of 0.1 ml of cell suspensions prepared in distilled water by a 1:100 dilution of the 0.5 McFarland suspension. After inoculation, the isolates resulted in incubation at 36 degrees C with clarithromycin at pH 7.4, and with the remaining agents at pH 6.6. The growth endpoints were visually read after 7-day and 10-day incubations for slowly growing nontuberculous mycobacteria, and after 3-day and 5-day incubations for rapidly growing mycobacteria, respectively. The reproducibility was evaluated with the five ATCC reference strains of nontuberculous mycobacteria. Of the 1,287 repeated tests of the four ATCC slowly growing strains(M. avium, M. intracellulare, M. kansasii and M. gordonae), 1,200(93.2%) of the MICs read after 7-day incubation fell within 3 log 2 dilutions. Also, a strain of rapidly growing mycobacteria, M. fortuitum, was repeatedly tested, and the reproducibility was estimated to be 93.3% after 3-day incubation. A total of 728 clinical isolates of nontuberculous mycobacteria comprising 14 species were tested against nine agents. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of rifampicin, levofloxacin, clarithromycin were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative means to determine MICs for nontuberculous mycobacteria in clinical laboratories.

Anti-Bacterial Agents↗

PubMed1999

Occurrence of nontuberculous mycobacteria in environmental samples.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).

Colony Count, Microbial↗