Slow-growing Mycobacterial Species Research Articles

Fluorogenic Probes of the Mycobacterial Membrane as Reporters of Antibiotic Action.

The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.

Assessing Boron-Pleuromutilin AN11251 for the Development of Antibacterial Agents.

Pleuromutilins are a group of antibiotics derived from the naturally occurring compound. The recent approval of lefamulin for both intravenous and oral doses in humans to treat community-acquired bacterial pneumonia has prompted investigations in modifying the structure to broaden the antibacterial spectrum, enhance the activity, and improve the pharmacokinetic properties. AN11251 is a C(14)-functionalized pleuromutilin with a boron-containing heterocycle substructure. It was demonstrated to be an anti-Wolbachia agent with therapeutic potential for Onchocerciasis and lymphatic filariasis. Here, the in vitro and in vivo PK parameters of AN11251 were measured including PPB, intrinsic clearance, half-life, systemic clearance, and volume of distribution. The results indicate that the benzoxaborole-modified pleuromutilin possesses good ADME and PK properties. AN11251 has potent activities against the Gram-positive bacterial pathogens tested, including various drug-resistant strains, and against the slow-growing mycobacterial species. Finally, we employed PK/PD modeling to predict the human dose for treatment of disease caused by Wolbachia, Gram-positive bacteria, or Mycobacterium tuberculosis, which might facilitate the further development of AN11251.

Complete Genome Sequence of Mycobacterium heckeshornense JCM 15655T, Closely Related to a Pathogenic Nontuberculous Mycobacterial Species, Mycobacterium xenopi.

Mycobacterium heckeshornense is a slow-growing mycobacterial species for which pathogenic features are unclear. Here, we report the complete genome sequence of an M. heckeshornense type strain. This sequence will provide essential information for future taxonomic and comparative genome studies of these mycobacteria.

Mycobacterium helveticum sp. nov., a novel slowly growing mycobacterial species associated with granulomatous lesions in adult swine.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with ‘ Mycobacterium avium subsp. hominissuis ’. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense , a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83T (=DSM 109965T= LMG 2019-02457T) is the type strain.

The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Functional Characterisation of Three O-methyltransferases Involved in the Biosynthesis of Phenolglycolipids in Mycobacterium tuberculosis

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

EmergingMycobacteriaspp. in Cooling Towers

To the Editor: The importance of nontuberculous mycobacteria (NTM) in various clinical situations recently has increased. Members of the Mycobacterium avium complex (MAC) cause a high percentage of infections in persons with acquired immunodeficiency syndrome. Some species are considered emerging pathogens, particularly those of the M. chelonae–M. abscessus group. They occur not only in immunocompromised persons but also in persons without predisposing conditions. Although sources of infection are considered to originate from the human environment, until now cooling towers were not clearly demonstrated to be one of these contamination sources, despite being suspected (1). Natural streams, groundwater, brook waters, and swamps already were reported to contain different species of NTM. Constructed environments, such as hospital hot water systems, aerosols from showers, ice machines, swimming pools, dental unit water, endoscopes, and bronchoscopes are other sources (2). Other studies have shown members of MAC in drinking-water distribution systems (3). A 1999 study in South Africa reported the presence of mycobacteria in cooling towers but reported no details about species (4). In 2003, cooling towers were reported to be a potential source of slow-growing mycobacterial species (1). Some mycobacterial species recently were shown to survive within amebae (5). We aimed to study the population of ameba-associated mycobacterial species in 3 cooling towers using a co-coculture method. These cooling towers (E, H, and O), located in downtown Paris, France, were sampled in May 2006. Water was taken in a sample point of the cooling circuit located just before the entrance of the tower basins. Because these cooling towers are regularly treated by oxidizing biocide BCDMH (1-bromo-3-chloro-5,5-dimethylhydantoin) to prevent development of Legionella spp., no Legionella spp. or Legionella-like amebal pathogens were isolated. Two liters of water samples were filtrated through 0.22-µm pore-sized filters that were injected onto amebal microplates as previously described (6) and incubated at 32°C. We screened amebal microplates by examination under inverted microscope and by Ziehl-Nielsen, Gram, and Gimenez staining. Positive wells were subcultured on axenic medium and incubated at 32°C for 10 days. Acid-fast isolates were identified by using partial rpoB gene amplification and sequencing with Myco-F and Myco-R primers (7). These primers did not allow amplification of partial rpoB gene for the 6 M. phocaicum strains. We thus used Myco-F/Myco-Rbon (5′-AGCGGCTGCTGGGTGATCAT-3′) primer pair for M. phocaicum (8). We compared these sequences with the rpoB gene sequence of Mycobacteria type strains available in the GeneBank database by using BLASTn on the NCBI website (www.ncbi.nlm.nih.gov). We observed bacteria growing in amebae. Subculture on axenic media led to polymicrobial cultures and allowed isolation of 33 mycobacterial strains (Table). All these strains were submitted to molecular identification. The 33 isolates corresponded to 5 mycobacterial species: M. fortuitum, M. conceptionense, M. chelonae, M. chimaera, and M. phocaicum (Table). Some of these mycobacteria already had been shown to survive in free-living amebae and to be implicated in human diseases, such as M. chelonae (5). The same author demonstrated recently that 26 environmental mycobacteria survived in the trophozoites and cysts of Acanthamoeba polyphaga (5). The recently described species M. phocaicum was isolated only in samples from humans and was associated with chronic pneumonia (9). However, the natural source of this species is still unknown. M. fortuitum was described as an opportunistic Mycobacterium associated with disseminated infections in a leukemia patient or in furunculosis after footbath in nail salons. M. conceptionense belongs to the M. fortuitum group and was isolated in a posttraumatic osteitis inflammation (10). M. chimaera belongs to MAC and has been isolated only in patients with pulmonary disorders but not from immunocompromised patients. The authors reported that the isolate showed unusually high virulence. We isolated this species for the first time in an environmental sample. It could belong to the transitory flora of the H cooling tower because it was isolated only once in our samples. Cooling towers already had been investigated for slow-growing mycobacteria (1), and our study showed they also can be an environmental source of rapidly growing NTM pathogens. In our procedure for amebal co-cultures, incubation cannot last >10 days because amebae do not survive longer in Page’s amebal saline buffer. Moreover, agar plates were incubated for 10 days only, which explains why we could recover only rapidly growing mycobacteria. The cooling towers, already known as a source of dissemination of Legionella spp., may disseminate mycobacteria in aerosols and be a previously nonrecognized source of infection. Table Identification of isolated nontuberculous mycobacteria according to their rpoB gene sequence similarity and GenBank accession numbers

BCG菌とヒト型結核菌の迅速鑑別にPCR法が有用であった胸部皮下膿瘍・肋骨骨髄炎の1小児例

Conventional polymerase chain reaction (PCR) used to identify mycobacterial species does not distinguish between Mycobacterium tuberculosis and M. bovis BCG, and several weeks or months may be needed to identify individual slow-growing Mycobacterial species. We report a 4-year-old girl who had subcutaneous abscess and sternal osteomyelitis after BCG vaccination at 4 month of age. We directly identified M. bovis BCG genome in the punctured abscess within a few days using PCR and PCR-restriction fragment length polymorphism. Such PCR is useful for rapidly diagnosing and managing of appropriate therapy in patients with infection due to M. bovis BCG.

Method for Rapid Identification and Differentiation of the Species of the Mycobacterium chelonae Complex Based on 16S-23S rRNA Gene Internal Transcribed Spacer PCR-Restriction Analysis

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

Mycobacterium lentiflavum, a recently identified slow-growing mycobacterial species: clinical significance in immunosuppressed cancer patients and summary of reported cases of infection

The clinical significance of Mycobacterium lentiflavum, a recently identified nontuberculous mycobacterium, remains uncertain, especially in immunosuppressed cancer patients. The records of all patients in whom M. lentiflavum was identified using a gene sequencing technique between January 2001 and December 2003 were reviewed. The mean age among 12 patients was 51+/-20 years, and 11 (92%) patients had a hematologic malignancy. Six of seven (86%) hematopoietic stem cell transplant recipients had received allogeneic donor grafts. Nine (75%) patients had predisposing risk factors for infection, seven (58%) had severe lymphocytopenia (<400 cells/microl), five (42%) were receiving systemic corticosteroid therapy, and three (25%) had acute graft-versus-host disease. Only 1 of the 12 (8%) patients had evidence of probable pulmonary M. lentiflavum infection. Six M. lentiflavum strains were initially misidentified as Mycobacterium simiae and Mycobacterium avium-intracellulare complex using traditional biochemical tests. Four M. lentiflavum isolates were tested for antimicrobial susceptibility; they were susceptible to isoniazid, ethambutol, clarithromycin, and amikacin, and resistant to rifampin. M. lentiflavum was not clinically significant, even in these severely immunosuppressed cancer patients.

Analysis of Mycobacterium species for the presence of a macrolide toxin, mycolactone.

Mycobacterium ulcerans is an environmental organism which is responsible for the disease Buruli ulcer, a necrotizing skin disease emerging in west Africa. M. ulcerans produces the polyketide-derived macrolide mycolactone, which is required for the immunosuppression and tissue damage which characterizes Buruli ulcer. We have extracted lipids from the cell envelope and culture filtrate from 52 isolates of Mycobacterium species, analyzed them with thin-layer chromatography, and tested them in a murine fibroblast cell line (L929) cytotoxicity assay to investigate whether these mycobacterial species produce mycolactone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast- and slow-growing mycobacterial species. Isolates tested included 16 uncharacterized, slow-growing, environmental mycobacterial species isolated from areas in which M. ulcerans infection is endemic. Although several strains of mycobacteria studied produced cytopathic lipids, none of these produced a phenotype on cultured cells consistent with that produced by mycolactone. Two mycobacterial species, M. scrofulaceum and M. kansasii, and eight of the environmental mycobacterial isolates contained cell-associated lipids cytopathic to fibroblasts at concentrations of 33 to 1,000 microg/ml. In contrast, mycolactone produces cytotoxicity at less than 2 ng/ml. Analysis of 16S rRNA sequences from the eight environmental isolates suggests that these are novel mycobacterial species. Results from these studies suggest that, although production of cytopathic lipids is relatively common among mycobacterial species, the production of mycolactone as a cell-associated or secreted molecule appears so far to be restricted to M. ulcerans.

Cooling towers--a potential environmental source of slow-growing mycobacterial species.

Over the last decade a rise in the frequency of disease caused by nontuberculous mycobacteria (NTM) has occurred, especially among AIDS patients. The lack of evidence for person-to-person transmission indicates the environment is a source of infection. The ecology and environmental sources of NTMs are poorly understood, and many pathogenic strains have not been observed outside of clinical cases. Several species of NTMs have been reported from treated water distribution systems; however, one type of manmade environment that has not been examined for mycobacteria is that of cooling towers of air-conditioning systems. Such environments not only harbor a variety of microbial species, they also disseminate them in aerosols. The present investigation examined nine cooling towers from various locations in the United States. Cooling tower water was concentrated, treated with cetylpyridinium chloride, and plated onto Middlebrook 7H10 agar supplemented with OADC and cycloheximide. Colonies presumed to be mycobacterial species were isolated and acid-fast stained. Identification was made by amplifying and sequencing 1450 bp fragments of the 16S rRNA gene in both directions, and comparing resulting sequences with those in GenBank. Results showed that at least 75% of tower samples contained NTMs, and most of the isolates closely matched known mycobacterial pathogens. Isolates most closely matched the following GenBank sequences: Mycobacterium intracellulare, M. szulgai, M. bohemicum, M. gordonae, M. nonchromogenicum, and M. n. sp. "Fuerth 1999." This is the first report of specific NTMs in cooling tower water, and the first report of M. n. sp. "Fuerth 1999" from any environmental sample. Although cooling towers have a relatively high pH, they may favor the growth and dissemination of such potential pathogens, and future epidemiologic investigations should consider cooling towers as possible environmental sources of mycobacteria.

Cooling Towers—A Potential Environmental Source of Slow-Growing Mycobacterial Species

Cooling Towers—A Potential Environmental Source of Slow-Growing Mycobacterial Species

Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.

Genome structure of mycobacteriophage D29: implications for phage evolution

Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.

Further studies of a new pathogenic mycobacterium (M. haemophilum sp. nov.)

Mycobacterium haemophilum is an acid-fast rod-shaped organism, originally isolated from deep subcutaneous granulomata of a patient with Hodgkin's disease. Like the other two mycobacterial skin-pathogens, M. ulcerans and M. marinum, M. haemophilum has a maximum temperature for growth below 37 degrees C. Mycobacterium haemophilum is distinguished from all other species examined by its requirement of haemin for growth and its complete lack of catalase activity. Extraneous catalase cannot replace haemin as a growth factor for this organism. Mycobacterium haemophilum can also be differentiated from other species by the patterns of electrophoresis of protein extracts and by gas-liquid chromatography of saponificated and methylated lipid extracts. A monospecific-agglutinating antiserum against M. haemophilum was obtained by adsorption of an immunoserum with M. intracellulare. A number of slow-growing mycobacterial species develop on monolayers of McCoy fibroblasts, and growth on these tissue cultures can be observed much earlier than on artificial media. Mycobacterium haemophilum is characterized by exclusively intracellular development.

In Vitro Properties of Mycobacterium lepraemurium Strain Keishicho

The morphologic, biochemical, and drug susceptibility characteristics of the Keishicho strain, supposedly a strain of Mycobacterium lepraemurium, are compared with these same characteristics of representative strains of other slow-growing mycobacterial species. The Keishicho strain is quite distinct from all other mycobacteria. Special media and very large inocula are needed for in vitro growth; growth is exceedingly slow. The inter-taxon similarity coefficients were low; the highest, only 63%, was to Mycobacterium tuberculosis.

AN IMMUNODIFFUSION ANALYSIS OF MYCO-BACTERIUM LEPRAEMURIUM MARCHOUX AND SOREL

SUMMARY Six strains of Mycobacterium lepraemurium Marchoux and Sorel have been subjected to immunodiffusion analysis. They were found to be identical with each other and to possess 12 demonstrable antigenic constituents. Five of these were antigens common to all mycobacteria, two were shared only with slow-growing mycobacterial species, three were amongst those antigens hitherto considered specific to M. avium and two were specific to M. lepraemurium alone. Thus it was concluded that M. lepraemurium is a member of the slow-growing group of mycobacteria with an exceptionally close serological relationship to M. avium.