Delving into the Characteristic Features of "Menace" Mycobacterium tuberculosis Homologs: A Structural Dynamics and Proteomics Perspectives.

The global increase in the morbidity/mortality rate of Mycobacterial infections, predominantly renascent tuberculosis, leprosy, and Buruli ulcers have become worrisome over the years. More challenging is the incidence of resistance mediated by mutant Mycobacterium strains against front-line antitubercular drugs. Homologous to all Mycobacteria species is the GlcNAc-6-phosphate deacetylase (NagA) which catalyzes essential amino sugars synthesis required for cell wall architecture, hence, metamorphosing into an important pharmacological target for curtailing virulence and drug-resistance. This study used integrated bioinformatics methods, MD simulations, and DynaMut and PolyPhen2 to; explore unique features, monitor dynamics, and analyze the functional impact of non-synonymous single-nucleotide polymorphisms of the six NagA of most ruinous Mycobacterium species; tuberculosis (Mtb), smegmatis (MS), marinum (MM), ulcerans, africanum, and microti respectively. This approach is essential for multi-targeting and could result in the identification of potential polypharmacological antitubercular compounds. Comparative sequential analyses revealed ≤ 50% of the overall structure, including the catalytic Asp267 and reactive Cys131, remained conserved. Interestingly, MS-NagA and MM-NagA possess unique hydrophobic isoleucine (Ile) residues at their active sites in contrast to leucine (Leu) found in other variants. More so, unique to the active sites of the NagA is a 'subunit loop' that covers the active site; probably crucial in binding (entry and exit) mechanisms of targeted NagA inhibitors. Relatively, nsSNP mutations exerted a destabilizing effect on the native NagA conformation. Structural and dynamical insights provided, basically pin-pointed the "Achilles' heel" explorable for the rational drug design of target-specific 'NagA' inhibitors potent against a wide range of mycobacterial diseases.

A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures.

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae. In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

Molecular characterization of environmental mycobacterial species from leprosy endemic tribal regions of North Purulia District, West Bengal.

Background: The aim of the present study was to isolate and characterize nontuberculous mycobacteria (NTM) on Lowenstein-Jensen media supplemented with glycerol or pyruvate on two different temperatures from soil samples from leprosy endemic tribal areas of Purulia. Methods: Mycobacterium leprae DNA was isolated from these samples followed by polymerase chain reaction (PCR) amplification using RLEP gene target specific to M. leprae. DNA was extracted from NTM cultures by lysis method. The presence of Mycobacterial DNA was confirmed by PCR using universal mycobacterial primer as 16S rRNA. NCBI nBlast was used for the authentication of NTMs, and phylogenetic tree was constructed using M. leprae and NTM species. Statistical Analysis Used: The percentile method and phylogenetic tree were used as stastical tool in this research article. Results: The rapid-growing mycobacteria (RGM) species, 4 (80%) was obtained more than that of slow growing mycobacteria (SGM) 1 (20%) supplemented on glycerol at 30°C followed by SGM species 8 (62%) were recovered more than RGM at 37°C. Similarly, SGM species 2 (100%) were recovered on supplemented with pyruvate at 30°C and no RGM growth when supplemented with pyruvate. Further, the recovery of RGM species 3 (60%) was better on supplemented with pyruvate than SGM species at 37°C. Mycobacterium timonense was first time isolated from Indian soil samples. Highest numbers of NTM were isolated from bathing place than washing and sitting places along with M. leprae PCR positivity. Phylogenetic tree showed a close genetic evolutionary association between Mycobacterium simiae and M. leprae in the leprosy endemic environment. Conclusion: Several NTM was isolated from soil of leprosy endemic area which might have role in susceptibility of leprosy. Phylogenetic tree revealed a closed association of M. simiae with M. leprae in the environment and might be maintaining the leprosy endemicity in north block of Purulia.

Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India.

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.

Molecular Detection and Differentiation of Mycobacterium Tuberculosis Complex and Non-tuberculous Mycobacterium in the Clinical Specimens by Real Time PCR.

Mycobacteria are subdivided into three groups: the Mycobacterium tuberculosis complex, the non-tuberculous mycobacteria called NTM or MOTT (Mycobacteria Other Than Tuberculosis) and Mycobacterium leprae. Over the past few decades, the incidence of infections caused by NTM has increased world wide. The differentiation of Mycobacterium tuberculosis complex from NTM is of primary importance for infection control and choice of antimicrobial therapy. However, there is so far no report in Bangladesh about the detection of NTM and hence differentiation of MTB and NTM. Neither acid-fast bacilli (AFB) staining nor histopathology can discriminate MTB and NTM. In order to detect and differentiate Mycobacterium tuberculosis complex and NTM we used commercially available LyteStar TB/NTM Real Time PCR kit (Altona Diagnostics, Germany) and analyzed 782 clinical specimens from tuberculosis suspected patients. We have found 49 MTB and 74 NTM positive samples from variety of clinical specimens such as sputum, bronchial lavages, body fluids, tissues, needle aspirates and swabs. Many of the PCR positive specimens were AFB negative on direct microscopic examination thus, indicating strong sensitivity of PCR than AFB staining. This is the first report in the country about detection of NTM and it warrants further elaborate investigation. Moreover, our results showed that multiplex real-time PCR assay is an effective sensitive tool for the rapid identification and differentiation of MTB and NTM directly from clinical specimens.

Genomic characterization of Nontuberculous Mycobacteria.

Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic.

UNLABELLED: This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCE: Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues.

Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.

Infections due to non-tuberculous mycobacteria (NTM).

The membership list of genus mycobacterium is ever expanding and it has grown to 95 in year 2003. While leprosy and tuberculosis are specific diseases caused by mycobacteria, other members are usually saprophytes but can be opportunistic and at times deadly pathogens. These other mycobacteria are referred to as atypical mycobacteria, non-tuberculous mycobacteria (NTM) or mycobacteria other than tubercle bacilli (MOTT). These organisms can produce localized disease in the lungs, lymph glands, skin, wounds or bone. Occasionally they may produce disseminated disease. Of the more than 90 known species of NTM, about one third have been associated with disease in humans. The species causing human disease are : Mycobacterium avium, M. intracellulare, M. kansasii, M. paratuberculosis, M. scrofulaceum, M. simiae, M. habana, M. interjectum, M. xenopi, M. heckeshornense, M. szulgai, M. fortuitum, M. immunogenum, M. chelonae, M. marinum, M. genavense, M. haemophilum, M. celatum, M. conspicuum, M. malmoense, M. ulcerans, M. smegmatis, M. wolinskyi, M. goodii, M. thermoresistible, M. neoaurum, M. vaccae, M.palustre, M. elephantis, M. bohemicam and M. septicum. Isolation of these mycobacteria from representative specimens and their rapid identification is very important as the treatment strategy for tuberculosis and other mycobacterioses is different. Several biochemical, chemical (lipid) and molecular techniques have been developed for rapid identification of these species. Along with suggestive clinical features, poor response to antitubercular treatment and repeated isolation of the organisms from the clinical specimens these techniques can help in establishing correct diagnosis. Further, many drugs like rifampicin, rifabutin, ethambutol, clofazimine, amikacin, new generation quinolones and macrolides effective against mycobacterial infections are available that can be used in appropriate combinations and dosage to treat these infections.

Histologic and genotypic characterization of a novel Mycobacterium species found in three cats.

Three cases of feline atypical mycobacteriosis from different geographical regions in North America were characterized by large clusters of filamentous bacteria visible on hematoxylin-and-eosin-stained tissue sections. PCR amplification demonstrated the presence of Mycobacterium-specific nucleic acid in samples of skin lesions from these cases. PCR-assisted cloning and DNA sequence analysis of a 541-bp length of the Mycobacterium 16S rRNA gene generated DNA sequences which were >95% identical, suggesting that the three isolates were closely related. Two of the sequences were 99% identical and may represent the same species. Alignment with comparable 16S rRNA gene sequences from 66 Mycobacterium species and partially characterized isolates highlighted similarities (>94%) with Mycobacterium bohemicum, Mycobacterium haemophilum, Mycobacterium ulcerans, Mycobacterium avium subsp. avium, and isolate IWGMT 90242. Parsimony analysis of sequence data suggested relatedness to M. leprae. Significant molecular genetic and pathobiological differences between these three similar isolates and other known species of mycobacteria suggested that the organisms may not have been described previously and that these cases may represent a new form of mycobacterial disease in cats. We suggest the term "Mycobacterium visibilis" to describe the organism from which the two nearly identical sequences were obtained.

Detection of mycobacterial DNA in formalin-fixed, paraffin-embedded tissue specimens by duplex polymerase chain reaction: application to histopathologic diagnosis.

Granuloma is a chronic inflammatory process associated with noninfectious agents or infectious diseases such as tuberculosis. Determination of the causative agent might be occasionally difficult in histopathologic sections. In this study, we examined 60 specimens of granuloma or inflammatory lesions that were originally diagnosed as 51 cases of granulomatous inflammation, 6 of leprosy, and 3 of atypical mycobacteriosis. The diagnoses in the last two categories were made both histologically and clinically. All of the sections and DNA were prepared from formalin-fixed, paraffin-embedded blocks. Histopathologic and immunohistochemical findings were compared with the results of duplex polymerase chain reaction (PCR) using two primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 240-bp DNA. Six samples of leprosy and three of atypical mycobacteriosis showed the 383-bp but not the 240-bp band. Among the 51 specimens of granulomatous inflammations, nine showed no band of even the beta-globin, the cases being excluded from this analysis. The 42 specimens of granulomatous inflammation were subdivided into three categories by PCR: (1) 383- and 240-bp positive; (2) 383-bp positive and 240-bp negative; and (3) both negative. Category 1 included 32 specimens (76.2%), being considered as tuberculosis. One specimen was classified into Category 2, indicating possible atypical mycobacterium. Category 3 included nine specimens, composed of five of sarcoidosis and four other agent-induced granulomas, when compared with histologic and clinical findings. These findings indicate that the PCR assay using DNA extracted from paraffin-embedded materials provides useful information to differentiate tuberculosis from other type of granulomas.

Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region.

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Cloning and expression of the Mycobacterium fortuitum superoxide dismutase gene.

In this paper we report the cloning, sequencing and expression of the superoxide dismutase (sod) gene from Mycobacterium fortuitum. A single gene was found to code for superoxide dismutase activity with its identity being confirmed by expression in M. aurum. The amino acid sequence was found to be similar to that of superoxide dismutases of several other origins. A region downstream of the sod gene also showed similarities to the corresponding sequences of the two main mycobacterial pathogens: M. leprae and M. tuberculosis. Analysis of enzymatic activity showed this enzyme in M. fortuitum required manganese as cofactor.

An improved method for the species-specific assessment of mycobacteria in routinely formalin-fixed and paraffin-embedded tissues.

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the beta-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.

Detection and characterization of atypical mycobacteria by the polymerase chain reaction.

The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, and Mycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, including M. kansasii and Mycobacterium leprae, in formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin-embedded tissue sections.

Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis.

We have cloned and sequenced the genes coding for the 85B antigen from M. bovis BCG and M. tuberculosis. Within this gene, the only difference in sequence between M. bovis BCG and M. tuberculosis corresponds, respectively, to a C-->T yielding a Leu-->Phe replacement at position 100 of the mature 85B protein. Therefore as we described previously for the 85A gene, there is also very little variation between these two species within the 85B gene.

Mycobacterial protein antigens: a compilation.

In response to recommendations from the Steering Committees responsible for co-ordination of World Health Organization programmes for research on the immunology of leprosy (IMMLEP) and tuberculosis (IMMTUB), a list was prepared summarizing the properties of mycobacterial proteins currently under investigation with respect to their immunological activities. After consultation with more than 40 laboratories world-wide this list was extended to form the compilation shown below and is intended to provide a comprehensive and convenient reference for future studies in this field.

Structure of pAL5000, a plasmid from M. fortuitum and its utilization in transformation of mycobacteria.

We have developed a gene cloning system for mycobacteria. Based on the nucleotide sequence determined for the M. fortuitum plasmid pAL5000, we have constructed an E. coli/mycobacteria shuttle vector. This vector, pAL8, is composed of pAL5000, pTZ19R (an E. coli plasmid) and a kanamycin resistance gene (from Tn903). We were unable to obtain viable kanamycin resistant pAL8 transformants of M. smegmatis using a PEG-mediated DNA uptake system, in spite of the fact that we could show efficient DNA uptake by transfection using the mycobacterial lytic phage D29. However, kanamycin resistant transformants of M. smegmatis or BCG could be obtained by electroporation. This plasmid cloning system provides a tool for studies of the expression of cloned genes (e.g. virulence) or epitopes in mycobacteria and allows the rational construction of recombinant BCG polyvalent vaccines.

Beta-lactamase production and biological characteristics in nitrosoguanidine induced Mycobacterium fortuitum mutants.

In order to elucidate the role of beta-lactamase in the resistance of M. fortuitum to beta-lactams, M. fortuitum ATCC 19542 and three mutants, strains D 316, D 319 and D 170 obtained from it by nitrosoguanidine treatment, were studied. Furthermore the kinetics of the beta-lactamase production during the bacterial growth and many biochemical and enzymatic characteristics of parent and mutant strains were investigated. Amoxicillin MICs well correlated with the beta-lactamase production in the high producer strains D 316 and D 319; on the contrary in strain D 170 a high MIC was joined with a moderate production of the enzyme showing that not only beta-lactamase but also other mechanisms can be effective in the resistance of M. fortuitum to beta-lactams. Clavulanic acid, an inhibitor of beta-lactamase, reduced MICs to amoxicillin in high and in low producer strains. The production of extracellular beta-lactamase occurred in a M. fortuitum mutant strain mainly during the stationary phase, indicating that a cell wall damage or initial autolysis could be responsible for the release of enzyme. Enzymatic and biochemical characteristics were not affected by nitrosoguanidine treatment except for nitrate test which showed only a weak positivity in high producer strains.