A laboratory pilot study of thermal decontamination of soils polluted by PCBs. Comparison with thermogravimetric analysis.

Real soils contaminated with a mixture of polychlorinated biphenyls were thermally decontaminated in a laboratory scale thermal desorption apparatus, under different operating conditions including two operating pressures (P= 0.01 and 0.1 MPa), different sample masses (80-320 g), carrier gas flow rates (0-30-280 Nl h(-1)) and two initial contamination levels. A standard European soil artificially contaminated with 4-chlorobiphenyl was decontaminated using a thermogravimetric analyser (TGA). The same parametric study as cited earlier was performed. With both techniques, the extent of decontamination was studied as a function of temperature during the heating programs (2-3 degrees C min(-1) for the thermal desorption apparatus; 5 degrees C min(-1) for TGA). Only a few differences were noticed between the two techniques. The decontamination starts when the melting temperature of the contaminant is reached (30 degrees C) and is complete before 350 degrees C. Thermogravimetric analysis which does not necessitate any chemical analysis appears to be a very attractive technique to investigate the feasibility of a decontamination and to quickly determine the best operating conditions.

Adsorption and pressure swing desorption of NOx in Na-Y zeolite: experiments and modeling.

Pressure swing NOx adsorption-desorption cycles were performed in the temperature range 200-350 degrees C using a fixed adsorbent bed of compressed Na-Y pellets and using a honeycomb coated with Na-Y powder. The experiments were performed using a synthetic gas mixture mimicking exhaust from a lean burn internal combustion engine. Na-Y zeolite coadsorbs NO and NO2 as N2O3, which in the regeneration were displaced by competitively adsorbed water molecules from a hydrated air stream. The performance of the fixed bed in these NOx adsorption and displacement desorption processes were modeled with a one-dimensional model. The kinetic and thermodynamic parameters from the fixed bed model were implemented in a model for the operation of the monolith. The experimental adsorption and desorption NOx concentration profiles in the monolith were reasonably well reproduced by the model. The water content of the flushing stream and the stripping gas flow rate are key process parameters. Technically, both parameters can be optimized in a valveless system with rotating honeycomb adsorbent comprising a NOx adsorption, a water injection and a NOx evacuation section.

RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different.

Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period.

Mycobacterium intracellulare as a cause of a recurrent granulomatous tenosynovitis of the hand.

We report a case of recurrent granulomatous tenosynovitis with M. intracellulare in a 55-year-old HIV negative diabetic woman. Identification of the causative agent further than belonging to the M. avium-intracellulare complex is provided by specific PCR-amplification of genomic DNA and sequencing of an hypervariable region within its 16S RNA gene. Sixteen months antibiotic regimen of rifabutin and clarithromycin led to a complete resolution of the tenosynovitis.

The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis.

We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis. These subunits, i.e. the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure. A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M. tuberculosis. Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters. Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters. By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M. tuberculosis. The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs. Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers. Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M. tuberculosis to many antibiotics. Interestingly, a transporter (absent in E. coli and in B. subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified. In comparison to E. coli and B. subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M. tuberculosis. This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients. The genes encoding the ABC transporters occupy about 2.5% of the genome of M. tuberculosis.

Listeria monocytogenes possesses adhesins for fibronectin.

Listeria monocytogenes is a gram-positive, nonsporulating, food-borne pathogen of humans and animals that is able to invade many eukaryotic cells. Several listerial surface components have been reported to interact with eukaryotic cell receptors, but the complete mechanism by which the bacteria interact with all of these cell types remains largely unknown. In this work, we found that L. monocytogenes binds to human fibronectin, a 450,000-Da dimeric glycoprotein found in body fluids, on the surface of cells and in an insoluble component of the extracellular matrix. The binding of fibronectin to L. monocytogenes was found to be saturable and dependent on proteinaceous receptors. Five fibronectin-binding proteins of 55.3, 48.6, 46.7, 42.4, and 26.8 kDa were identified. The 55.3-kDa protein was proved to be present at the bacterial cell surface. The binding of L. monocytogenes to fibronectin adds to the number of molecules to which the bacterium is able to adhere and emphasizes the complexity of host-pathogen interactions.

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family.

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.

Thermogravimetric study of thermal decontamination of soils polluted by hexachlorobenzene, 4-chlorobiphenyl, naphthalene, or n-decane.

To determine decontamination behavior as affected by temperature, shallow beds of a clay-rich, a calcerous, and a sedimentary soil, artificially polluted with hexachlorobenzene, 4-chlorobiphenyl, naphthalene, or n-decane, were separately heated at 5 degrees C min-1 in a thermogravimetric analyzer. Temperatures for deep cleaning of the calcerous and the sedimentary soil increased with increasing boiling point (bp) of the aromatic contaminants, but removal efficiencies still approached 100% well below the bp. Decontamination rates were therefore modelled according to a pollutant evaporation-diffusion transport model. For the calcerous and sedimentary soils, this model reasonably correlated removal of roughly the first 2/3 of the naphthalene, but gave only fair predictions for hexachlorobenzene and 4-chlorobiphenyl. It was necessary to heat the clay soil above the aromatics bp to achieve high decontamination efficiencies. Weight loss data imply that for temperatures from near ambient to as much as 150 degrees C, interactions of each aromatic with the clay soil, or its decomposition products, result in lower net volatilization of the contaminated vs. neat clay. A similar effect was observed in heating calcerous soil polluted with hexachlorobenzene from near ambient to about 140 degrees C. Decontamination mechanisms remain to be established, although the higher temperatures needed to remove aromatics from the clay may reflect a more prominent role for surface desorption than evaporation. This would be consistent with our estimates that the clay can accommodate all of the initial pollutant loadings within a single surface monolayer, whereas the calcerous and sedimentary soils cannot.

Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR.

Listeria monocytogenes strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing L. monocytogenes strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints, L. monocytogenes strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of L. monocytogenes was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for L. monocytogenes typing.

PCR assay fails to detect molluscum contagiosum virus-related sequences in AIDS-related Kaposi's sarcoma.

Previous PCR-based studies have demonstrated the presence of various viral DNA or RNA sequences in Kaposi's sarcoma (KS) tissues. To date, only human herpesvirus 8 (HHV-8) DNA sequences are found consistently in KS. The putative role of this agent in KS pathogenesis remains, however, to be determined; HHV-8 could infect populations endemically and could be reactivated in patients with KS. A close association between AIDS-related KS and molluscum contagiosum occurrence was found and this study was conducted primarily to search for the presence of molluscum contagiosum virus DNA sequences in KS. Frozen KS samples were examined for the presence of both HHV-8 and molluscum contagiosum virus DNA sequences by PCR. Despite a high rate of co-infection, no molluscum contagiosum virus (MCV) DNA sequence could be found in the KS samples whereas HHV-8 was uniformly detected. These results suggest that the high prevalence of MCV in AIDS patients with KS relies on a mode of transmission common for HHV-8 and molluscum contagiosum virus rather than on a multiviral etiology of KS. They may also indicate a particular susceptibility of the host to viral reactivation. If this is so, the failure to detect MCV DNA sequences in KS tissues by PCR indicates that locally produced or released cyotokines are not involved in the latter process.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis.

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phas of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.

Sporadic case of listeriosis associated with the consumption of a Listeria monocytogenes-contaminated 'Camembert' cheese.

Listeria monocytogenes is an intracellular gram-positive organism responsible for severe infections in both humans and animals. Whereas the food-borne transmission of listeriosis was demonstrated in several outbreaks, most cases of listeriosis occur sporadically and are rarely linked with consumption of contaminated foods. In this paper a case of septicaemia with L. monocytogenes in a 73-year-old immunocompromised man is described. Evidence for the association of this case of listeriosis with the consumption of a contaminated 'Camembert' cheese is provided by serotyping, esterase typing, DNA macrorestriction patterns analysis and level of virulence of the isolated strains for mice.

Cloning and characterization of transcriptional promoters from Bacillus subtilis phage 2C.

Phage 2C is a Bacillus subtilis lytic phage, whose genome contains hydroxymethyluracil in place of thymine. To isolate promoters of early phage genes involved in the take-over of cellular metabolism, 2C DNA libraries were constructed in promoter-probe plasmids replicating in Escherichia coli and B. subtilis. Four different 2C DNA fragments strongly expressed reporter genes in E. coli but not in B. subtilis. All fragments originated from unique sequences of the genome and not from its terminal redundancies. One fragment was sequenced. Despite the presence of an sigma-A-RNA polymerase binding site upstream of the transcriptional initiation site of a 2C early gene, this fragment did not promote transcription in B. subtilis.

Serotyping and esterase typing for analysis of Listeria monocytogenes populations recovered from foodstuffs and from human patients with listeriosis in Belgium.

Listeria monocytogenes strains isolated in Belgium from different foodstuffs and in sporadic cases of human listeriosis were analyzed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human patients with listeriosis were further studied by esterase typing. The twenty esterase patterns defined were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C and the pathogenicity level of strains in immunocompromised mice could not explain the unequal distribution of esterase types. The discrimination index of esterase typing (DI = 0.868) was compared with that of serotyping (DI = 0.666) and with that of the two combined methods (DI = 0.899).

Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing.

Esterases from Listeria monocytogenes strains isolated from cheeses were analyzed by starch gel electrophoresis. Five esterases, numbered from EST 1 to EST 5 in order of decreasing anodal migration, were identified. The EST 1, EST 3, EST 4, and EST 5 set was most active toward alpha-naphthyl propionate, while EST 2 was most active toward alpha-naphthyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed in the population. Twelve esterase patterns were defined and used to subdivide serotypes.

Paratuberculosis.

Paratuberculosis (Johne's disease) is a chronic, wasting, widespread mycobacteriosis of ruminants. It involves extensive mycobacterial shedding, which accounts for the high contagiousness, and ends with a fatal enteritis. Decreases in weight, milk production, and fertility produce severe economic loss. The DNA of the etiological agent (Mycobacterium paratuberculosis) has a base composition (66 to 67% G+C) within the range of that of mycobacteria (62 to 70% G+C), a size (4.4 x 10(6) to 4.7 x 10(6) bp) larger than that of most pathogenic mycobacteria (2.0 x 10(6) to 4.2 x 10(6) bp), and a high relatedness (> 90%) to Mycobacterium avium DNA. However, the DNAs of the two organisms can be distinguished by restriction fragment length polymorphism analysis. M. paratuberculosis genes coding for a transposase, a cell wall-associated protein (P34), and two heat shock proteins have been cloned and sequenced. Nucleic acid probes (two of which are species specific) are used, after PCR amplification, for M. paratuberculosis identification in stools and milk. As in leprosy, with disease progression, cellular immune reactions decrease and humoral immune reactions increase. Cutaneous testing with sensitins, lymphocyte proliferation assays, and cytokine tests are used to monitor cellular immune reactions in paratuberculosis, but these tests lack specificity. Complement fixation, immunodiffusion, and enzymometric tests based on antibodies to M. paratuberculosis extracts, to mycobacterial antigen complex A36, to glycolipids, and to proteins help identify affected cattle but are not species specific. The carboxyl-terminal portion of the 34-kDa cell wall-associated A36 protein (P34) carries species-specific B-cell epitopes and is the basis for an enzyme-linked immunosorbent assay. Diagnostic tests for paratuberculosis are also used in Crohn's disease, a chronic human ileitis mimicking Johne's disease, in which isolates identified as M. paratuberculosis have been found.

Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle.

The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.

Mycobacterium paratuberculosis and Escherichia coli share common antigenic determinants.

Paratuberculosis (Johne's disease) is a chronic enteritis of ruminants, which is due to infection by Mycobacterium paratuberculosis. By comparative analysis of Western blots of M. paratuberculosis components incubated with sera from paratuberculous cows (either pre-absorbed or not on an Escherichia coli sonicate), we have shown the presence of cross-reactive antigens between M. paratuberculosis and E. coli. Components in the range of 22 to 75 kDa are recognized by sera from paratuberculous cows, but only some components in the range of 20 to 40 kDa are endowed with specific B-cell epitopes to M. paratuberculosis with respect to E. coli. Cross-reactions were further stressed by the fact that rabbit immunoglobulins to E. coli recognized some ten M. paratuberculosis components (in the range of 45 to 66 kDa). The importance of cross-reactivity between the two bacterial species was evaluated by enzyme-linked immunosorbent assay (ELISA) using soluble sonic-extract components from M. paratuberculosis as antigens. Pre-absorption of series of bovine sera with E. coli resulted in the diminution of ELISA mean optical density readings by 16.1% and 62.0% for paratuberculous or healthy animals, respectively.