A resistant (R) clinical isolate of Alcaligenes denitrificans subsp. xylosoxydans was recovered from CSF during treatment including piperacillin. This variant selected in vivo, and a second variant obtained in vitro from the initially susceptible (S) strain, both exhibited resistance to penicillins (ticarcillin, piperacillin) and cephalosporins, but remained susceptible to latamoxef and imipenem. Clavulanate (2 mg/L) restored the susceptibility of the two R-variants to penicillins. A beta-lactamase of pI 9.5 was detected in both S and R strains, but overproduction was observed only in the in-vivo and in-vitro R-variants. This inducible beta-lactamase hydrolysed benzylpenicillin, cephalothin and cephaloridine efficiently, but amoxycillin, ticarcillin and cefoperazone were only moderate substrates. The enzyme was inhibited by clavulanate, cloxacillin and imipenem (IC50 between 3 and 9 mM), but not by aztreonam and chloride ions (1 mM). Resistance to beta-lactams was not transferable by conjugation to Escherichia coli or Pseudomonas aeruginosa, and DNA agarose gel electrophoresis indicated that no plasmid was present in the isolates. Restriction patterns of chromosomal DNA isolated from the S and R isolates were similar after digestion by NotI and HindIII.

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A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km. The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms. The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase. As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid. By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector. Efficiency of the transposition process was 0.58%. This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.

The ability of Bacillus strains to degrade geosmin, a musty off-flavor component present in superficial waters, led some authors to postulate the existence of a Bacillus exoenzyme: the “geosminase”. Such an enzyme, as an immobilized form onto a granular material, would be of a great interest to remove geosmin from water. This prospect prompted us to look for geosminase in Bacillus cultures. An aqueous solution of pure geosmin was added to Bacillus culture supernatants from exponential or stationary phases, and to a whole culture induced for 24 hours with 0.06 ppm of geosmin. Geosmin was determinated by GLC as a function of incubation time. No evidence of enzymatic activity was obtained, but an important adsorption of geosmin onto glass vessels was observed at neutral pH (6.7–7.5). Geosmin was recovered from glass by elution with methanol. Thus great care must be taken before assuming a biological origin to geosmin disappearance from superficial waters. However, preliminary results indicate that a “true” geosminase can be detected in Actinomycete lysates. The characterization and the purification of this enzyme are actually in progress.