Nontuberculous mycobacterial species and Mycobacterium tuberculosis complex coinfection in patients with pulmonary tuberculosis in Dr. Soetomo Hospital, Surabaya, Indonesia.

OBJECTIVE/BACKGROUND: The aim of this study was to analyze the detection of nontuberculous mycobacterial (NTM) species derived from sputum specimens of pulmonary tuberculosis (TB) suspects. Increasing prevalence and incidence of pulmonary infection by NTM species have widely been reported in several countries with geographical variation. MATERIALS AND METHODS: Between January 2014 and September 2015, sputum specimens from chronic pulmonary TB suspect patients were analyzed. Laboratory examination of mycobacteria was conducted in the TB laboratory, Department of Clinical Microbiology, Dr. Soetomo Hospital, Surabaya. Detection and identification of mycobacteria were performed by the standard culture method using the BACTEC MGIT 960 system (BD) and Lowenstein-Jensen medium. Identification of positive Mycobacterium tuberculosis complex (MTBC) was based on positive acid-fast bacilli microscopic smear, positive niacin accumulation, and positive TB Ag MPT 64 test results (SD Bioline). If the growth of positive cultures and acid-fast bacilli microscopic smear was positive, but niacin accumulation and TB Ag MPT 64 (SD Bioline) results were negative, then the isolates were categorized as NTM species. MTBC isolates were also tested for their sensitivity toward first-line anti-TB drugs, using isoniazid, rifampin, ethambutol, and streptomycin. RESULTS: From 2440 sputum specimens of pulmonary TB suspect patients, 459 isolates (18.81%) were detected as MTBC and 141 (5.78%) as NTM species. CONCLUSION: From the analyzed sputum specimens, 18.81% were detected as MTBC and 5.78% as NTM species. Each pulmonary TB suspect patient needed clinical settings to suspect causative agents of MTBC and/or NTM species; clinicians have to understand the local epidemiological data for the evaluation of causes of lung infection to determine appropriate therapy.

Ultrastructural morphologic changes in mycobacterial biofilm in different extreme condition.

The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 °C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 °C as compared to 30 °C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 °C as compared to 37 and 42 °C. Mycobacterium tuberculosis developed strong biofilm at 37 °C and no biofilm at 30 and 42 °C in Sauton's media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton's medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multi-layered, three-dimensional structure.

Phagocytosis and cytokine response to rough and smooth colony variants of Mycobacterium abscessus by human peripheral blood mononuclear cells.

Mycobacterium abscessus is a non-tuberculous mycobacteria able to cause opportunistic infections in selected patient groups. During the last decades it has emerged as a cause of chronic pulmonary infection in patients with cystic fibrosis (CF). M. abscessus strains exhibit either smooth or rough colony morphology. Strains exhibiting the rough phenotype more often cause pulmonary infections in CF patients than did the smooth ones. Here, we examined phagocytosis and production of cytokines by human peripheral blood mononuclear cells, in response to M. abscessus strains with smooth and rough colony phenotype. The rough isolates all formed multicellular cords, similar to what is observed in Mycobacterium tuberculosis. Monocytes were generally unable to internalize these rough cord isolates, in contrast with the smooth ones. Furthermore, the rough M. abscessus strains induced a distinct cytokine profile differing from that induced by the smooth ones. Rough isolates induced significantly less IL-10 and tumour necrosis factor compared to smooth strains, but more IL-1ß. Both varieties induced equal amounts of IFN-γ, IL-17, IL-23, IL-6, IL-8 and equally little IL-12. The ability to withstand phagocytosis might be a virulence factor contributing to the capacity of rough M. abscessus strains to give persistent pulmonary infections.

Enfermedad diseminada por Mycobacterium szulgai con compromiso pulmonar y ganglionar en un paciente con sida / Pulmonary infection and dissemination to cervical lymph nodes for Mycobacterium szulgai in AIDS patient

Mycobacterium szulgai es una MNT descrita como nueva especie patógena para el hombre en 1972 por Marks y colaboradores. El aislamiento de esta especie, una de las 115 descritas en el género Mycobacterium, no es frecuente en los seres humanos. M. szulgai es responsable de menos del 1 % de todos los aislamientos de MNT en los seres humanos; por esta razón, su aislamiento siempre se considera como agente patógeno. La presentación clínica de la enfermedad pulmonar producida por este microorganismo suele ser muy similar a la tuberculosis; el diagnóstico se presumirá cuando el tratamiento antituberculoso inicial no produzca la respuesta esperada. Asimismo, también se aísla ocasionalmente en afecciones osteoarticulares, compromiso cutáneo o ganglionar. El objetivo de este estudio fue describir el primer caso en nuestro país de infección respiratoria con diseminación ganglionar por M. szulgai en un paciente cubano con sida.

Enfermedad diseminada por Mycobacterium szulgai con compromiso pulmonar y ganglionar en un paciente con sida / Pulmonary infection and dissemination to cervical lymph nodes for Mycobacterium szulgai in AIDS patient

Mycobacterium szulgai es una MNT descrita como nueva especie patógena para el hombre en 1972 por Marks y colaboradores. El aislamiento de esta especie, una de las 115 descritas en el género Mycobacterium, no es frecuente en los seres humanos. M. szulgai es responsable de menos del 1 % de todos los aislamientos de MNT en los seres humanos; por esta razón, su aislamiento siempre se considera como agente patógeno. La presentación clínica de la enfermedad pulmonar producida por este microorganismo suele ser muy similar a la tuberculosis; el diagnóstico se presumirá cuando el tratamiento antituberculoso inicial no produzca la respuesta esperada. Asimismo, también se aísla ocasionalmente en afecciones osteoarticulares, compromiso cutáneo o ganglionar. El objetivo de este estudio fue describir el primer caso en nuestro país de infección respiratoria con diseminación ganglionar por M. szulgai en un paciente cubano con sida.(AU)

Preliminary characterization of a Mycobacterium abscessus mutant in human and murine models of infection.

The ability to persist in the host after the establishment of infection is an important virulence determinant for mycobacteria. Mycobacterium abscessus is a rapidly growing mycobacterial species which causes a variety of clinical syndromes in humans. We have obtained a rough, wild-type human clinical isolate of M. abscessus (M. abscessus-R) and a smooth, attenuated mutant (M. abscessus-S) which spontaneously dissociated from the clinical isolate. We have found that M. abscessus-R is able to persist and multiply in a murine pulmonary infection model in contrast to M. abscessus-S, which is rapidly cleared. To understand the basis for this difference, we characterized the behavior of these variants in human tissue culture models of infection. M. abscessus-R is able to persist and multiply in human monocytes, while M. abscessus-S is deficient in this ability. Both of these variants are phagocytized by human monocytes. M. abscessus-R resides in a phagosome typical for pathogenic mycobacteria with a tightly adherent phagosomal membrane. In contrast, M. abscessus-S resides in a "loose" phagosome with the phagosomal membrane separated from the bacterial cell wall. Both M. abscessus variants also have distinctive growth patterns in a recently described fibroblast-mycobacterium microcolony assay, with M. abscessus-R exhibiting growth characteristics similar to those previously reported for virulent M. tuberculosis and M. abscessus-S exhibiting growth characteristics similar to those previously reported for avirulent M. tuberculosis. In both the monocyte infection assay and the murine pulmonary infection model, numerous infected mononuclear phagocyte aggregates develop at sites of M. abscessus-R infection, but are absent with M. abscessus-S infection. We conclude that a mutation has occurred in the M. abscessus-S variant which has altered the ability of this organism to persist and multiply in host cells and that this may be related to the phenotypic changes we have observed in our tissue culture models of infection.

[Utility of the direct culture examination (Ziehl-Neelsen) using the Bactec system for the presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi, and Mycobacterium kansasii]. / Valor del examen directo (Ziehl-Neelsen) del cultivo en sistema Bactec para la identificación presuntiva de: Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi y Mycobacterium kansasii.

BACKGROUND: The usefulness of the microscopic examination of Bactec 12B and 13A growth medium as a method for the possible identification of M. tuberculosis complex, M. avium complex, M. xenopi, and M. kansasii was performed out to guide the selection of different genetic identification probes and, in the case of M. xenopi, the selection of the temperature of subcultures incubation. METHODS: Upon detection of an index of growth greater than 100 in Bactec tubes, staining was performed by the Ziehl-Neelsen technique. On the basis of the morphology observed, the possible identification was performed by genetic probes. Subcultures were used for definitive identification. RESULTS: Three hundred forty-four positive samples were studied by radiometric technique. A total of 190 strains were identified as M. tuberculosis, 88 strains as M. avium-intracellulare (MAI), 33 strains as M. xenopi, 14 strains as M. kansasii and 19 strains were identified as: M. gordonae (10), unpigmented rapid growth microbacteria (7), and M. simiae (2). Sensitivity, specificity, positive predictive value, and negative predictive value were 97.9%, 95.4%, 96.4%, and 97.3%, respectively for M. tuberculosis complex, 84.0%, 99.2%, 97.3% 94.7% for M. avium complex; 63.6%, 98.3%, 80.7%, 96.2% for M. xenopi; 35.7%, 98.1%, 45.5% 97.2% for M. kansasii. CONCLUSIONS: The morphology of M. tuberculosis complex examined in the radiometric system in useful to differentiate this species from other microbacteria (MOTT), allowing the selection of specific probe used. Within the MOTT, M. avium complex also has morphological characteristics which are useful for its differentiation, the morphology usually described for the remaining species was frequently not observed.

Preservation of surface lipids and determination of ultrastructure of Mycobacterium kansasii by freeze-substitution.

The cell wall architecture of a slowly growing mycobacterium, Mycobacterium kansasii, was examined by freeze-substitution following growth in vitro. Freeze-substituted bacteria were marked by the presence of an electron-translucent space (or electron-transparent zone [ETZ] described by previous workers [T. Yamamoto, M. Nishiura, N. Harada, and T. Imaeda, Int. J. Lepr. 26:111-114, 1958]) surrounding the majority of cells. At least two morphotypes of mycobacteria were revealed by freeze-substitution. In the first, a relatively thin (11 +/- 2.3 to 3.5 +/- 3.1 nm), uniform ETZ surrounded intact cells which contained cytoplasm filled with well-stained ribosomes and a DNA nucleoid distributed throughout the cell. The second morphotype consisted of a small proportion of organisms that were distorted in shape and were surrounded by a much thicker (59 +/- 2.6 to 198 +/- 2.5 nm) ETZ in areas of the cell which appeared to have retracted from the space it had originally occupied, leaving depressions in the ETZ. The lipid nature of the ETZ was demonstrated because cells were devoid of an ETZ when organisms were freeze-substituted in the absence of osmium tetroxide in the substitution medium or treated with neutral lipid solvents (acetone or ethanol) before freeze-substitution. Moreover, thin-layer chromatography of acetone or ethanol extracts obtained from solvent-treated cells identified a lipid component which corresponded to the M. kansasii-specific phenolic glycolipid. In contrast, negligible amounts of glycolipids were detected in extracts obtained from control HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer-treated cells, and these cells retained an ETZ. These results demonstrate that species-specific phenolic glycolipids are essential components in the architecture of the M. kansasii ETZ. Furthermore, we show that freeze-substitution is a reliable technique for the retention and precise preservation of lipid-containing polymers in the mycobacterial cell wall.

Physical organization of lipids in the cell wall of Mycobacterium chelonae.

Mycobacterial cell wall functions as an effective permeability barrier, making these bacteria resistant to most antibacterial agents. It has been assumed that this low permeability was due to the presence of a large amount of unusual lipids in the cell wall, but it was not known how these lipids are able to produce such an exceptional barrier. We report here the first experimental evidence on the physical arrangement of these lipids based on X-ray diffraction studies of purified Mycobacterium chelonae cell wall, a result suggesting that the hydrocarbon chains of the cell-wall lipids are arranged predominantly in a direction perpendicular to the cell wall surface, probably producing an asymmetric bilayer structure.

Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols.

The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.

The role of coccoid ultrafine forms of mycobacteria in the transmission of the mycobacterial infection.

The present study is devoted to the variability of the mycobacterial population in nature by transformation of typical mycobacteria into ultrafine forms with a dense cellular membrane in the organism of mosquitos and of their larvae. Mosquitos of the genus Aedes are known to be mechanical carriers of bacterial infections. Most of the infectious diseases are characteristic of rural districts and have the character of natural foci. An attempt has been made to determine the possibility of the transmission of the mycobacterial infection through the stings of bloodsucking mosquitos of the genus Aedes. It is shown that transmission of the mycobacterial infection is possible through the stage of coccoid ultrafine mycobacteria detected in the mosquito larvae of different stages. The reversion of the coccoid forms of mycobacteria into the typical rod-shaped ones is possible in separate cases and occurs at later stages of the mosquito larvae development. The study may be of scientific and practical interest both in medicine and agricultural practice.

[The surfaces of colonies of Mycobacterium species studied in the scanning electron microscope]. / Issledovanie poverkhnostei kolonii nekotorykh vidov mikobakterii v skaniruiushchem élektronnom mikroskope.

The study of the morphology of the surface of several mycobacterial species differing in their pathogenicity by the methods of scanning and transmission electron microscopy has revealed that cells in such colonies and micrococlonies are associated and form one common layer of medium electron density. The cells in this layer are sharply outlined. The cover on the surface of mycobacterial colonies, revealed in this investigation, ensures the stability of mycobacteria in the environment and their resistance to the action of various chemical and physical factors.

Buruli ulcer: clinical pathological study of 23 patients in Lambaréné, Gabon.

Within one year 23 patients in Lambaréné (Gabon) were diagnosed as having a Buruli ulcer (advanced cases with undermined margins). The majority of the ulcers were found in children; with the exception of one ulcer on the thorax, all ulcerations were localised at the extremities. The mode of transmission could not be clarified; extensive water tests for Mycobacterium ulcerans were negative. Observed under an electron microscope, M. ulcerans has a similar structure as other mycobacteria. Exudative inflammatory processes predominate in the ulcer floor. The small blood vessels play a considerable role in the pathological process. The mycobacteria are present in this layer, below this there is widespread necrosis of adipose tissue. The margin of the ulcer displays a granulomatous inflammatory reaction. The cytological characteristics of the mesenchymal reaction of a Buruli ulcer are described with the aid of electron microscope photographs.

The pathology of Mycobacterium ulcerans infection.

The pathology of cutaneous ulcers resulting from Mycobacterium ulcerans infection is reviewed. Initial infection causes ulceration with necrosis of the dermis and a septate panniculitis in subcutaneous fat. There is little cellular reaction despite the presence of large numbers of organisms. Recurrent or persistent infection produces a granulomatous reaction with epithelioid macrophages, variable numbers of giant cells of the Langhans type, and relatively few organisms. This type of reaction is associated with more successful treatment of the disease and appears analogous to the tuberculoid form of leprosy.

Freeze-fracture observations of the cell walls and peribacillary substances of various mycobacteria.

Ultrastructure of the cell wall and peribacillary substances of various mycobacteria (32 strains of 18 species) grown in vitro was studied by a freeze-fracture technique. Peribacillary substances differed in shape among species and even among strains of the same species, and were classified into five types: (1) amorphous substances; (2) multi-layered sheaths with no filamentous units; (3) structures composed of filaments of 2-4 nm diameter, which were further classified into three subtypes according to the arrangement of the filaments; (4) helical fibres; and (5) single fibres, or networks of fibrous structures, with no visible substructures. No strains revealed peribacillary structures resembling those of uncultivable Mycobacterium leprae. These results have implications for the mechanism of freeze-fracturing in mycobacterial walls.

Isolation in high frequency of rough variants of Mycobacterium intracellulare lacking C-mycoside glycopeptidolipid antigens.

Rough variants of serovars from the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex were isolated in high frequency from pellicle growth of the wild-type strains. Rough morphology could be correlated with the lack of an outer cell wall sheath and its constituent C-mycoside glycopeptidolipids of both the serologically active and inactive types.