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We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.

One hundred ninety-five individual vancomycin-resistant Enterococcus faecium (VRE) isolates from five upstate New York hospitals were studied for antimicrobial susceptibilities to LY333328, quinupristin-dalfopristin, teicoplanin, ampicillin, and gentamicin. LY333328 was the most active antibiotic against VRE. The effect of media and methods on the antibacterial activity of LY333328, its synergy with ampicillin, and the postantibiotic effects (PAE) of LY333328 and ampicillin were evaluated. In microdilution tests, the MIC of LY333328 at which 90% of the isolates were inhibited (MIC 90 ) was 2 g/ml in Mueller-Hinton II (MH II) broth and 1 g/ ml in brain heart infusion (BHI) broth. In contrast, on MH II agar the MIC 90 was 4 g/ml and on BHI agar it was >16 g/ml. Bactericidal activity was observed for most strains at concentrations from 8 to >133 times the MIC of the tube macrodilution in MH II broth. A bactericidal effect of LY333328 plus ampicillin was demonstrated in time-kill studies, but there was great strain-to-strain variability. By the MH II agar dilution method, bacteristatic synergy (defined as a fractional inhibitory concentration of <0.5) with LY333328 and ampicillin was demonstrated for 61% of the strains tested. Under similar conditions, there was synergy with LY333328 and quinupristin-dalfopristin or gentamicin for 27 and 15% of the strains tested, respectively. The PAE of LY333328 was prolonged (23.0 h at 10 times the MIC). However, 50% normal pooled human serum decreased the PAE to 12.2 h at 10 times the MIC. Test conditions and media had a considerable effect on VRE susceptibilities to LY333328. The prolonged PAE of LY333328, a potent new bactericidal glycopeptide, and its synergy with ampicillin in a large proportion of strains suggest that further evaluation of this drug in pharmacokinetic studies and experimental infections, including those with VRE, is warranted.

We investigated the antistaphylococcal activity of daptomycin, vancomycin, oxacillin, gentamicin, and rifampin in human monocyte-derived macrophages. Compared with vancomycin and oxacillin, daptomycin had the most rapid and greatest antibacterial activity, but that of oxacillin was most sustained. The combination of daptomycin, gentamicin, and rifampin was most effective intracellularly, while daptomycin plus gentamicin and the three-drug combination were most effective extracellularly, completely eliminating viable Staphylococcus aureus.

The conjugative TOL plasmid (75 Mdal), specifying biodegradation of xylenes, toluene, and trimethylbenzene derivatives, undergoes dissociation in Pseudomonas aeruginosa PAO to a nonconjugative TOL* plasmid (28 Mdal) and a transfer plasmid termed TOLA (48 Mdal). The A large number of plasmid genes that specify drug or mercury resistance are now known to be located in discrete DNA segments that can dissociate from the plasmids and insert themselves into other replicons (1-3). In many cases, transposition of such DNA segments has been demonstrated to occur by terminus site-specific recA-independent recombination systems that may be facilitated by the presence of insertion sequences (IS) as either direct or inverted repeats at the termini (4,5). Such IS elements have also been implicated in the recombination of F plasmids with chromosomal genes to yield F' plasmids (6) or in the association-dissociation phenomena observed with some plasmids such as R100 and R6-5 (7, 8). Most of the transposable DNA segments characterized so far involve drug resistance, and this has been interpreted as an efficient molecular mechanism for the evolution of new drug resistance characters on plasmids and their rapid proliferation among microorganisms. In this communication, we present evidence that suggests that some hydrocarbon degradative genes that occur naturally as part of conjugative plasmids in Pseudomownas putida strains can be dissociated from the conjugative plasmids to form nonconjugative plasmids and can readily form plasmid cointegrates during transfer with sex-factor plasmids. In addition, such degradative genes can undergo further translocation from the plasmid cointegrates onto other replicons and promote gene inactivation at or near the site of insertion. MATERIALS AND METHODSBacterial Strains. Details of the maintenance and growth conditions of P. putida strains harboring various plasmids have been described (9, 10). The relevant genotypic characteristics and the nature of plasmids borne by these strains are given in (AC 839) were grown overnight in L broth. Aliquots (0.1 ml) were then inoculated into 2 ml of fresh L broth and grown for 2 hr at 320. The cells were centrifuged and plated on minimal m-toluate (0.1%) plates supplemented with tryptophan (50 Ag/ml) and streptomycin (500 Ag/ml). After incubation for 5 days at 320, the colonies were checked for resistance to other relevant antibiotics. For transposition of salicylate degradative genes, SAL+RP4::TOL*+ P. putida AC 842 strain was mated with AC 10, and exconjugants were selected on glucose minimal plates containing methionine (50,gg/ml) and carbenicillin (5 mg/ml). The carbenicillin-resistant (CI9) colonies were then replica-plated on minimal salicylate/methionine and toluate/methionine plates. Exconjugants that were Sal+Cbr but Tol-Kms appeared at a frequency of 10-7. Such colonies were tested for transferability of Cbr and SAL genes to E. coli recipients to ensure the presence of RP4 replication and transfer genes in the transposition derivatives.

We investigated the antistaphylococcal activities of daptomycin, gentamicin, and rifampin against two Staphylococcus aureus strains and their stable small-colony variants, singly and in combination, in human monocyte-derived macrophages and in broth. Intracellularly, the three-drug combination and two-drug combinations with rifampin were most effective. Extracellularly, daptomycin, daptomycin plus gentamicin, gentamicin plus rifampin, and the three-drug combination had similar activities.

Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance.