Increased whiB7 expression and antibiotic resistance in Mycobacterium chelonae carrying two prophages.

BACKGROUND: The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although integrated viral genomes, called prophage, are linked to increased antibiotic resistance in some bacterial species, we know little of their role in mycobacterial drug resistance. RESULTS: We present here for the first time, evidence of increased antibiotic resistance and expression of intrinsic antibiotic resistance genes in a strain of Mycobacterium chelonae carrying prophage. Strains carrying the prophage McProf demonstrated increased resistance to amikacin. Resistance in these strains was further enhanced by exposure to sub-inhibitory concentrations of the antibiotic, acivicin, or by the presence of a second prophage, BPs. Increased expression of the virulence gene, whiB7, was observed in strains carrying both prophages, BPs and McProf, relative to strains carrying a single prophage or no prophages. CONCLUSIONS: This study provides evidence that prophage alter expression of important mycobacterial intrinsic antibiotic resistance genes and additionally offers insight into the role prophage may play in mycobacterial adaptation to stress.

Diagnosis of Mycobacterium abscessus/chelonae complex cutaneous infection: Correlation of tissue culture and skin biopsy.

Mycobacterium abscessus and M. chelonae belong to the rapid-growing nontuberculous mycobacteria (NTM) group, which are defined by their ability to form visible colonies on agar within 7 days of subculture. Cutaneous infections by this complex show a heterogeneous clinical presentation with varied histopathologic findings. However, the presence of vacuoles in many specimens has been reported as a specific histologic finding. Herein, we correlate the histopathology of patients with tissue-culture positive M. abscessus/M. chelonae complex in order to identify features that may prompt a rapid categorization of the infectious etiology. The cohort includes 33 skin punch biopsy specimens from 28 patients who had associated positive tissue cultures. The most frequent clinical presentation was a single or multiple nodule. Twenty-seven specimens (81.81%) were found to have vacuoles. The observation of certain histologic features (ie, polymorphonuclear microabscesses and epithelioid granuloma formation) should raise the possibility of infection by NTM. In addition to these findings, we believe the presence of vacuoles in the dermal and subcutaneous inflammation should raise suspicion for NTM.

Gene replacement in Mycobacterium chelonae: application to the construction of porin knock-out mutants.

Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.

Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard.

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M. fortuitum infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.

Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability.

The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.

Comparison of methods for Identification of Mycobacterium abscessus and M. chelonae isolates.

Mycobacterium abscessus and Mycobacterium chelonae are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either M. abscessus or M. chelonae that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be M. abscessus and 29 were identified as M. chelonae. Eight isolates originally submitted as M. chelonae were identified as M. abscessus, and one isolate submitted as M. abscessus was found to be M. chelonae. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of M. abscessus exhibited resistance to tobramycin (MIC of 8 to > or =16 microg/ml), while all isolates of M. chelonae were susceptible to this drug (MIC of < or = 4 microg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of M. abscessus and M. chelonae.

Binding of alpha2-laminins by pathogenic and non-pathogenic mycobacteria and adherence to Schwann cells.

The ability of Mycobacterium leprae to specifically bind alpha2-laminins of Schwann cells has been described recently as being an important property of the leprosy bacillus, which could explain the neural tropism of M. leprae. Therefore, the extent of the expression of alpha2-laminin-binding properties among mycobacteria was investigated. In an ELISA-based assay, all three species of Mycobacterium tested (M. tuberculosis, M. chelonae and M. smegmatis) expressed laminin-binding capacity, suggesting that the ability to bind alpha2-laminins is conserved within the genus Mycobacterium. This report also demonstrated that not only M. leprae but all the mycobacterial species tested readily interacted with the ST88-14 cells, a human schwannoma cell line, and that the addition of soluble alpha2-laminins significantly increased their adherence to these cells. These results failed to demonstrate the presence in M. leprae of a unique system based on alpha2-laminins for adherence to Schwann cells.

Reduced glutaraldehyde susceptibility in Mycobacterium chelonae associated with altered cell wall polysaccharides.

Glutaraldehyde-resistant Mycobacterium chelonae have been isolated from endoscope washer disinfectors and endoscope rinse water. The mechanism of glutaraldehyde resistance is not well understood. Two spontaneous, glutaraldehyde-resistant mutants of the sensitive type strain, NCTC 946, were investigated. The colony morphology of the two mutants differed from that of the the type strain: colonies of the former were dry and waxy whereas those of the latter were smooth and shiny. Increased resistance to glutaraldehyde of the mutants was matched by small increases in the MICs of rifampicin and ethambutol but not isoniazid. Both mutants showed increased surface hydrophobicity. No changes were identified in the extractable fatty acids or the mycolic acid components of the cell wall but a reduction in each of the resistant strains in the arabinogalactan/arabinomannan portion of the cell wall was detected.

[Postoperative infection outbreak of M. chelonae subsp. abscessus].

OBJECTIVE: To study the pathogen of postoperative infection outbreak in a hospital in Shenzhen. METHODS: According to the Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis published by Chinese Antituberculosis Association, Bergey's Manual of Determinative Bacteriology (ninth edition), and the Clinical Bacteriology Examination & Development, 97 wound exudates were cultivated by the traditional bacteriologic method. The strains were identified with three standard strains of M. chelonae subsp. chelonae, M. chelonae subsp. abscessus and M. fortuitum as controls. RESULTS: 26 fast-acid positive and fast growth nontuberculous mycobacterium were isolated from 97 exudates, then 41 strains including other 15 strains which were collected from the hospital were identified as M. chelonae subsp. abscessus. CONCLUSION: The outbreak of the postoperative infection happened in the hospital was caused by M. chelonae subsp. abscessus.

Strategies used by pathogenic and nonpathogenic mycobacteria to synthesize rRNA.

One rRNA operon of all mycobacteria studied so far is located downstream from a gene thought to code for the enzyme UDP-N-acetylglucosamine carboxyvinyl transferase (UNAcGCT), which is important to cell wall synthesis. This operon has been designated rrnAf for fast-growing mycobacteria and rrnAs for slow growers. We have investigated the upstream sequences and promoter activities of rrnA operons of typical fast growers which also possess a second rrn (rrnBf) operon and of the rrnA operons of the fast growers Mycobacterium abscessus and Mycobacterium chelonae, which each have a single rrn operon per genome. These fast growers have a common strategy for increasing the efficiency of transcription of their rrnA operons, thereby increasing the cells' potential for ribosome synthesis. This strategy involves the use of multiple (three to five) promoters which may have arisen through successive duplication events. Thus we have identified a hypervariable multiple promoter region (HMPR) located between the UNAcGCT gene and the 16S rRNA coding region. Two promoters, P1 and PCL1, appear to play pivotal roles in mycobacterial rRNA synthesis; they are present in all of the species examined and are the only promoters used for rRNA synthesis by the pathogenic slow growers. P1 is located within the coding region of the UNAcGCT gene, and PCL1 has a characteristic sequence that is related to but distinct from that of the additional promoters. In fast-growing species, P1 and PCL1 produce less than 10% of rRNA transcripts, so the additional promoters found in the HMPR are important in increasing the potential for rRNA synthesis during rapid growth. In contrast, rrnB operons appear to be regulated by a single promoter; because less divergence has taken place, rrnB appears to be younger than rrnA.

Clinical significance, biochemical features, and susceptibility patterns of sporadic isolates of the Mycobacterium chelonae-like organism.

Mycobacterium chelonae-like organisms are nonpigmented rapidly growing mycobacteria whose clinical significance is unknown. We evaluated 87 sporadic isolates encountered in a clinical laboratory. Most isolates (62%) were respiratory; only 2 of 54 (4%) (both from patients with AIDS) were clinically significant. Among 33 nonrespiratory isolates, 20 of 33 (or 61%) were clinically significant. Clinical diseases included posttraumatic wound infections and catheter-related sepsis. Routine biochemical features included growth inhibition by 5% NaCl (100%), a smooth colony morphology (94%), positive 3-day arylsulfatase reaction (84%), no color or a light tan color on iron uptake (100%), and variable nitrate reduction (45%). Additional characteristics that helped to separate this group from M. chelonae and Mycobacterium abscessus were susceptibility to cephalothin (90%) and ciprofloxacin (100%), utilization of mannitol (94%) and citrate (83%) as carbon sources, and unique patterns of mycolic acid esters by high-performance liquid chromatography. This group was quite drug susceptible, with 100% of isolates inhibited by amikacin, imipenem, cefoxitin, cefmetazole, and the newer quinolones ciprofloxacin and ofloxacin. Three examples of this group, including a proposed type strain, have been deposited in the American Type Culture Collection.

Characterization of the channel formed by the mycobacterial porin in lipid bilayer membranes. Demonstration of voltage gating and of negative point charges at the channel mouth.

We studied the channel formed by the mycobacterial porin from the cell wall of Mycobacterium chelonae (Trias, J., Jarlier, V., and Benz, R. (1992) Science 258, 1479-1481) by reconstituting the mycobacterial porin and cell wall extracts in lipid bilayer membranes. The channel exhibited two different states in lipid bilayer membranes at 10 mV of applied voltage. One was characterized by a steplike appearance while the other showed a fast, voltage-dependent, flickering behavior between a closed and an open state. The channel was voltage-gated, and starting at 40 mV of applied voltage the mycobacterial porin channel switched to a closed configuration in an asymmetric fashion. The channel was cation-selective and had 2.5-point negative charges at both sides of the channel. Identical channels were observed when membranes were reconstituted with cell wall extracts, suggesting that there is only one porin species in the mycobacterial cell wall.

Porins in the cell wall of mycobacteria.

The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics. Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.