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A total of 10 metallo-␤-lactamase-producing isolates of six different species, including Brevundimonas diminuta (n ‫؍‬ 3), Rhizobium radiobacter (n ‫؍‬ 2), Pseudomonas monteilii (n ‫؍‬ 1), Pseudomonas aeruginosa (n ‫؍‬ 2), Ochrobactrum anthropi (n ‫؍‬ 1), and Enterobacter ludwigii (n ‫؍‬ 1), were detected in the sewage water of a hospital. The presence of bla VIM-13 associated with a Tn1721-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, which produced VIM-2), and in two of them (R. radiobacter), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-␤-lactamase genes.Metallo-␤-lactamases (MBLs) have emerged worldwide as a major source of acquired broad-spectrum ␤-lactam resistance. They hydrolyze virtually all classes of ␤-lactams (except monobactams), including carbapenems, which often represent the last option for the treatment of infections with multidrugresistant Gram-negative bacteria.There are two dominant types of transferable MBLs among clinical isolates, IMP and VIM (12). Most of the IMP-and VIM-type MBL genes are present as gene cassettes inserted into integrons located on the chromosome or on plasmids. These integrons may be associated with transposon-like structures which may contribute to their variable location and spread (12). Interestingly, these MBL genes have been found almost exclusively in the hospital setting and the role of nonclinical habitats as a reservoir for bacteria that carry these acquired resistance determinants has been poorly investigated (9,10).In this study, we evaluated the presence of MBL-producing bacteria in the sewage water of Son Llatzer Hospital (Mallorca, Spain) in order to obtain epidemiological data which could complement the results from a previous survey that investigated the incidence of MBL-producing Pseudomonas aeruginosa strains in this hospital (4).For this purpose, duplicates of up to six 10-fold dilutions of 1 liter of sewage water collected 20 m downstream of the hospital wastewater discharge site were plated on three different media specially designed for the selection of pseudomonads (Gould S1 agar [1], King B agar [6], and cetrimide agar [Merck, Darmstadt, Germany]) containing 30 g/ml ceftazidime (Combinopharm, Madrid, Spain). A total of 37 bacterial isolates corresponding to different colony morphologies were collected using this strategy and tested for the presence of MBL using the MBL Etest according to the manufacturer's instructions (bioMérieux, Marcy l'Etoile, France). Only 16 isolates were positive by this test.Although these 16 MBL Etest-positive isolates exhibited different colony morphologies, their identification by 16S rRNA gene amplification and sequencing as previously described (13) showed that they belonged to only eight different species, including Brevundimonas diminuta, Rhizobium radiobacter, Pseudomonas monteilii, P. aeruginosa, Ochrobactrum anthropi, Enterobacter ludwigii, Acinetobacter johnsonii, a...

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg2+. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (Kobs = 1.2 (±0.3) × 105 M−1) is tighter than that of MSK (2.9 (±1.0) × 104 M−1) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.

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