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[Identification of Mycobacteriaceae species based on the hsp-65 gene polymorphism analysis by PCR - RFLP].

The polymorphism of the short fragment of the heat shock protein 65 encoding gene was evaluated by the PCR - RFLP technique described by Telenti and further developed by Devallois for identification of mycobacterial species in routine laboratory work. We analysed 58 strains representing 25 different mycobacterial species (24 reference strains and 34 clinical isolates). The results obtained by PCR-RFLP and HPLC identification techniques were highly concordant The results were compatible for 87,5% (21 / 24) reference strains and for 97,1% (33/34) clinical isolates. The PCR - RFLP method allowed for accurate identification mycobacterial species, especially pathogenic strains. Restriction patterns obtained for 25 species of Mycobacteriaceae genus could help in constructing the data base and algorithms used in routine laboratory practice.

Algorithms↗

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of Mycobacteriaceae.

The extracts having diverse immunostimulating activities were obtained as a water-phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol-water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram-negative bacteria and amphipathic substances from gram-positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons alpha/beta and gamma in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti-lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS-Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid A's), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.

Adjuvants, Immunologic↗

[Systematics, differentiation, and detection of bacterial infections-- the family Mycobacteriaceae].

Comparative 16S rRNA sequencing allows to infer natural relationships among bacteria, to characterize and identify microorganisms at a molecular level and to develop DNA probes specific at any desired taxonomic level (e.g. family, genus, species). Probes targeted at ribosomal RNA are suitable for in situ hybridization of whole, intact bacterial cells as well as in polymerase-chain-reaction techniques for sensitive detection and identification of bacteria. Comparative 16S rRNA sequencing provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. Certain growth characteristics, i.e. thermotolerance and growth rate correspond to natural relationships among the mycobacteria. However, the phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotochromogenic and nonphotochromogenic mycobacteria. The use of oligonucleotides targeted at highly or semi-conserved regions within the 16S rRNA molecule allows a universal procedure for amplification and rapid sequence determination of 16S rDNA-gene fragments from any virtually bacterial organism. This method of amplification of 16S rDNA-gene fragments was used to identify a novel, uncultured pathogen and opens new perspectives for other infectious diseases of unknown cause.

Base Sequence↗

In vitro antimicrobial activity and penetration rate into sputum of gatifloxacin, a novel 6-fluoro-8-methoxy quinolone, and its therapeutic efficacy in respiratory infections.

The in vitro antimicrobial activity of gatifloxacin against clinical isolates of pathogenic bacteria was evaluated. Minimum inhibitory concentrations of gatifloxacin, ofloxacin, ciprofloxacin, tosufloxacin, sparfloxacin, and rifampicin against 20 strains each of methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, and Pseudomonas aeruginosa, 18 strains of Enterobacter cloacae, 15 strains each of Streptococcus Pneumoniae and Moraxella catarrhalis, 12 strains of Haemophilus influenzae, 18 strains of Mycobacterium intracellulare, and 22 strains each of Mycobacterium tuberculosis and Mycobacterium avium were determined. The minimum inhibitory concentrations90's of gatifloxacin against the above species were 0.12, 8, <==0.06, 0.5, 2, 2, <==0.06, 0.39, 0.05, 0.013, 2, 0.5, and 4 &mgr;g/ml, respectively. Gatifloxacin was four times as active as ofloxacin against methicillin-susceptible and -resistant Staphylococcus aureus, and as active as ofloxacin against Enterobacteriaceae and Pseudomonas aeruginosa. Gatifloxacin was as active as tosufloxacin and sparfloxacin against Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. The antimycobacterial activity of gatifloxacin was similar to that of sparfloxacin. Five patients with chronic respiratory infections and one patient with acute pneumonia received 100-400 mg/day of gatifloxacin orally for 5-12 days (mean, 8.17 days). The clinical effects were excellent in one patient and good in five. One strain of Haemophilus influenzae was eradicated and one strain of Pseudomonas aeruginosa persisted after therapy. Adverse reactions were mild and improved after completion of therapy. In one patient with chronic bronchitis, the maximum sputum concentrations 2-4 h after oral administration of 150 mg of gatifloxacin on days 1 and 6 were 0.88 and 1.45 &mgr;g/ml, respectively, and in serum the values were 0.84 and 1.24 &mgr;g/ml, respectively. Thus it was found that gatifloxacin possesses potent activity against respiratory pathogens (including Mycobacteriaceae), and shows good penetration rate into sputum, and that it can be used as the drug of first choice in the treatment of respiratory tract infections.

Journal Article↗

[Diagnosis of mycobacteria].

Diagnostic methods of mycobacterial species can lead to significant problems. This article aims at guiding through the great number of mycobacteriaceae, differentiating between atypical and tuberculous mycobacterials, answering diagnostic questions, therefore making early specific therapy possible.

Bacteriological Techniques↗

A DNA probe for the detection of Mycobacterium paratuberculosis.

A genomic library of DNA extracted from Mycobacterium paratuberculosis was constructed in the expression vector lambda gt 11. The library was screened by plaque hybridization with labelled M. paratuberculosis genomic DNA as probe. Strongly hybridizing plaques were isolated and their DNA extracted and characterised for M. paratuberculosis specificity by hybridization to DNA from other Mycobacteriaceae. A clone was obtained which was specific for M. paratuberculosis. DNA from this clone could detect 7 ng M. paratuberculosis DNA.

Animals↗

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis.

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.

Animals↗

Systemic visceral talc granulomatosis associated with miliary tuberculosis in a drug addict.

Light and scanning electron microscopic study of tissues obtained at biopsy and at autopsy from a 64-year-old male drug addict revealed the presence of foreign body granulomas associated with birefringent crystals in the lungs, liver, bone marrow, spleen, and lymph nodes. Caseating granulomas associated with Mycobacteriaceae tuberculosis were also present. Energy dispersive X-ray analysis identified the crystals as talc. Minute talc crystals were also visualized in urine sediment, by scanning electron microscopy and identified by energy dispersive X-ray analysis.

Granuloma↗

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis.

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.

Animals↗

Identification and characterization of IS-like elements in Mycobacterium gordonae.

A new insertion sequence, IS1512, from Mycobacterium gordonae was cloned and sequenced. This element is present in up to 10 copies which provides a high diversity for restriction fragment length polymorphism analysis. We have also identified truncated IS1512-like elements, including a truncated IS1512 and another truncated insertion sequence which displays homology with IS1512 and was designated IS1511. Sequences homologous to the previously described transposon Tn554 are inserted into these truncated insertion sequences. Insertion loci of IS1511/IS1512 are shown to be highly related to those described for IS256-like elements in other species. Alignment of the putative transposases from Rhodococcus and Mycobacterium suggests these insertion sequences may form a distinct closely homologous subclass within the IS256 family. Analysis comparing phylogenetic divergence of these elements with that of 16S rRNA and superoxide dismutase genes suggests horizontal transfer of a IS1511/IS1512 precursor into M. gordonae, and the occurrence of horizontal transfer between Mycobacteriaceae and Rhodococcaceae.

Actinomyces↗

ABUNDANT MICROORGANISM IN SOIL.

The presence in soil of large numbers of a catalase-negative, microaerophilic, coccoid microorganism was demonstrated. Use of media of high nutrient value, without incorporation of inhibitors, and growth in the absence of antagonistic microorganisms were utilized to isolate this organism from soil dilutions greater than those providing growth by other means. The organism described does not grow on soil extract agars and is missed by conventional counting techniques for soil organisms. On the basis of morphological and growth characteristics, this organism appears to have at least some taxonomic relationships to the families Actinomycetaceae and Mycobacteriaceae. It is proposed that this organism makes up much of the coccoid microflora of soil as observed by light and ultraviolet fluorescence microscopy.

Actinomyces↗

Rapid lysis technique for mycobacterial species.

The lysis of mycobacterial cells typically has been difficult and time-consuming. We report a method for the physical rupture of Mycobacterium paratuberculosis and several other members of the family Mycobacteriaceae using a Mini-Beadbeater cell disrupter (Biospec Products; Bartlesville, Okla.) and zirconium beads, a process which yields DNA and RNA of high molecular weight and in greater quantity than that obtained by rupture in a Ribi pressure cell.

Animals↗

Comparison of 16S ribosomal RNA genes in Clavibacter michiganensis subspecies with other coryneform bacteria.

Nearly complete sequences (97-99%) of the 16S rRNA genes were determined for type strains of Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. sepedonicus, and Clavibacter michiganensis subsp. nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that the C. michiganensis subsp. shared relatively high homology with the 16S rRNA gene of Clavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G+C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of the Clavibacter michiganensis subsp., Clavibacter xyli, Arthrobacter globiformis, Arthrobacter simplex, and Frankia sp.; another cluster consisted of members of the corynebacteria-mycobacteria-nocardia (CMN) group of Mycobacteriaceae including Tsukamurella paurometabolum; and Propionibacterium freudenreichii alone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.

Actinomycetales↗