Comparative evaluation of the Oxoid Salmonella Rapid Test with three other rapid salmonella methods

Stable auxotrophic mutants were isolated by N-methyl-N'-nitro-N-nitrosoguanidine treatment of Mycobacterium fortuitum NIHJ 1615, M. smegmatis NIHJ 1628 and M. vaccae NIHJ 1637. The number of stable mutants obtained were 0.17, 0.46 and 0.02% of the surviving mutagenized cells screened for the three species, respectively. Mutants differed from their parents in a single nutritional requirement except in the case of SM29, a mutant of M. fortuitum , and SM33, a mutant of M. smegmatis , which differed from their parents in two auxotrophic traits.     

Numerical classification of rapidly growing nonphotochromogenic mycobacteria

Numerical analysis of 211 strains of rapidly growing, nonphotochromogenic mycobacteria was carried out by using 116 characters. New species reported after 1981 and not yet compared with known species by numerical classification were included. At the level of 90% of the matching coefficient, the following species could be differentiated from each other and were shown as distinct clusters: Mycobacterium pulveris, M. moriokaense, M. chitae, M. diernhoferi, M. porcinum, M. fortuitum, M. chelonae subspecies chelonae, M. chelonae subspecies abscessus, M. smegmatis, M. agri, M. fallax, M. parafortuitum, M. fortuitum subspecies acetamidolyticum. Four taxa, M. fortuitum, M. porcinum, M. chelonae subsp. chelonae, and M. chelonae subsp. abscessus, were regarded as distinct. However, these were combined into one large cluster at a level of 90% of the matching coefficient and, therefore, were regarded as forming one series or complex. M. fortuitum subsp. acetamidolyticum was reported as a subspecies of M. fortuitum, but this was differentiated clearly from the other species including M. fortuitum.     

Classification of Mycobacterium farcinogenes and Mycobacterium senegalense by immunodiffusion and thin-layer chromatography of long-chain components

Comparative immunodiffusion studies and thin-layer chromatographic analyses of whole-organism acid methanolysates were performed on 37 strains of Mycobacterium farcinogenes, Mycobacterium senegalense and Nocardia farcinica. The latter were clearly distinguished from the mycobacteria in containing a single mycolic acid methyl ester and showing more precipitinogens with nocardial than with mycobacterial and rhodococcal reference systems. The distribution of precipitinogens showed that M. farcinogenes and M. senegalense were very closely related and that both showed a greater affinity to Mycobacterium fortuitum than to any of the other established species of Mycobacterium tested. The complex pattern of alpha-mycolates and characteristic polar mycolates found in both M. farcinogenes and M. senegalense has only previously been found in M. fortuitum and Mycobacterium smegmatis.     

PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2 ')-Ic gene from Mycobacterium tuberculosis and the aac(2 ')-Id gene from Mycobacterium smegmatis

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis , which has re-emerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2 ' )-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum . The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2 ' )-Ic gene from M. tuberculosis H37Rv and the aac(2 ' )-Id gene from M. smegmatis mc²155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2 ' )-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2 ' )-Ic and aac(2 ' )-Id genes has been studied in M. smegmatis and only aac(2 ' )-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'- N -acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2 ' )-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc²155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.     

Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria

The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Physicochemical Characterization of Tween 80-Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Tween 80-hydrolyzing esterases produced by various species of rapidly growing mycobacteria were partially purified from sonicated cell lysates by diethylaminoethyl (DEAE) cellulose and subsequent Sephadex G-150 column chromatographies. The amount of the esterase produced per gram of bacterial cells varied markedly with each species. Mycobacterium smegmatis, M. chelonei, and M. phlei were high producers and M. chitae and M. diernhoferi were low producers of Tween-hydrolyzing esterase. The resistance of each mycobacterial strain to oleic acid correlated well with their esterase-producing ability. All the esterases studied were adsorbed on DEAE cellulose in 50 mM Tris-HCl buffer (pH 7.5), indicating that they are acidic proteins. Esterases of M. smegmatis, M. chitae, M. fortuitum, and M. phlei were eluted from DEAE at high concentrations (0.11–0.18 m) of ammonium sulfate, while those of M. parafortuitum and M. diernhoferi were eluted at lower concentrations (0.05–0.08 m). With Sephadex G-150 gel filtration, all esterases were shown to have similar molecular weights (36,000 to 58,000). On the basis of heat-stability and trypsin- or chymotrypsin-sensitivity, these esterases were divided into three groups: one was heat-stable and protease-sensitive (M. smegmatis and M. fotuitum), another was heat-labile and protease-resistant (M. chelonei), and the other was the intermediate of the above two groups (M. diernhoferi).     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Functional analysis of pAL5000 plasmid in Mycobacterium fortuitum.

Four of the five open reading frames (ORFs) present in Myobacterium fortuitum pAL5000 plasmid (ORF1, ORF3, ORF4, and ORF5) are dispensable for replication in M. fortuitum. However, two additional ORFs (ORF1 and ORF5) were necessary for replication in a Myobacterium smegmatis heterologous host containing an efficient plasmid transformation mutation.     

Effect of lysozyme on mycobacteria

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.     

Does aminoglycoside-acetyltransferase in rapidly growing mycobacteria have a metabolic function in addition to aminoglycoside inactivation?

All the rapidly growing mycobacteria tested, Mycobacterium fortuitum complex, M. smegmatis, M. phlei, and M. vaccae, contained one of two characteristics, but were different from previously recognized aminoglycoside-acetyltransferases. The acetylation reaction of both the enzymes from M. fortuitum and Pseudomonas aeruginosa (3-N-acetyltransferase-III) with radiolabeled acetyl coenzyme A was inhibited severely by oxalacetate. It was suggested that the inhibitory effect of oxalacetate is due to the condensation reaction between oxalacetate and acetyl coenzyme A resulting in the generation of citrate.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献   

Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450.

Nine bacterial strains that grew on morpholine and pyrrolidine as sole carbon, nitrogen, and energy sources were isolated from three different environments with no known morpholine contamination. One of these strains could also degrade piperidine. These bacteria were identified as Mycobacterium strains. A phylogenetic analysis based on the partial 16S rDNA sequences indicated that the isolated strains clustered within the fast growing group of mycobacteria. When the above-mentioned cyclic amines were used as growth substrates, the synthesis of a soluble cytochrome P450 was induced in all these bacteria. Other laboratory strains, Mycobacterium fortuitum and Mycobacterium smegmatis mc(2)155, were tested for their abilities to degrade morpholine. Neither of them degraded morpholine but could use pyrrolidine and piperidine. The growth of M. fortuitum and M. smegmatis mc(2)155 on these compounds involved a soluble cytochrome P450, suggesting that mycobacterial strains are naturally able to use pyrrolidine and have developed a similar enzymatic pathway to metabolize this amine.     

An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1.

A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.     

Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads

A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli. First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E. coli recA mutant cells. Secondly, plasmid DNA was isolated from recombinant E. coli cells and analysed. Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M. bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA. This protocol was also amenable to other mycobacterial species such as M. avium, M. fortuitum and M. tuberculosis H37Ra. Plasmid recovery from the recombinant M. bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method.     

Utilization of siderophores from mycobacteria by Staphylococcus

The ability of iron utilizing by means of siderophores (extracellular exochelins and cell-associated mycobactins) produced by mycobacteria (7 stains) by 24 staphylococcal strains was investigated. The mycobacterial donor strains belonged to rapid growing species: M. fortuitum, M. smegmatis, M. aurum, M. vaccae, M. chitae, M. phlei, M. parafortuitum. The utilization of mycobacterial siderophores was studied on agar or liquid media in which minimally effective concentrations of ethylene diamine di-ortho-hydroxyphenyl acetic acid (EDDA) were used to inhibit indicator staphylococcal strains. Mycobacterial siderophores (exochelins or mycobactins) reversed the inhibition of the majority (22/24) staphylococcal strains. Most of strains utilized exochelins from M. phlei as well as mycobactins from M. parafortuitum and M. chitae.     

The Electrophoretical Analysis of Esterase and Catalase and Its Use in Taxonomical Studies of Mycobacteria

The zymogram patterns of esterases and catalases of mycobacterial strains were studied using the thin layer agar electrophoresis. Though there were some variations, Mycobacterium hominis, M. bovis, M. kansasii, M. fortuitum, M. runyonii, M. avium, M. phlei and M. smegmatis seemed to show species-specific patterns consisting of 2 to 6 esterase bands and one or more catalase bands. The patterns of scotochromogens and nonchromogens were rather variable.     

Mycobactins as chemotaxonomic characters for some rapidly growing mycobacteria

Thirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, 'M. diernhoferi', M. duvalii, M. flavescens, M. fortuitum, M. gadium, 'M. gallinarum', M. neoaurum, M. parafortuitum, 'M. peregrinum', M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and 'M. kanazawa' failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and 'M. peregrinum') formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, 'M. diernhoferi', M. vaccae and M. parafortuitum). The mycobactin from 'M. gallinarum' was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.