Natural antibiotic resistance of bacteria isolated from larvae of the oil fly, Helaeomyia petrolei.

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 microg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.

A two-part energy burden imposed by growth of Enterobacter cloacae and Escherichia coli in sodium dodecyl sulfate.

Enterobacter cloacae, like most enteric bacteria, can grow in the presence of 10% sodium dodecyl sulfate (SDS). The bacteria tolerate the detergent and do not metabolize it. In a defined glucose-salts medium the growth rate remained unchanged (G = 55 min) as the detergent concentration was increased from 0 to 10% SDS. However, growth in SDS exhibited a two-part energy dependence. In part 1, the SDS-grown cells underwent rapid lysis when they ran out of energy. Cells that had entered stationary phase owing to carbon limitation lysed, while those that had entered owing to nitrogen or phosphorus limitation did not. We attribute part 1 of the energy dependence to SDS as a detergent. In part 2, the cells grown in 5 or 10% SDS exhibited longer lag periods, potassium accumulation, decreased cell yields, and higher oxygen consumption. The higher oxygen consumption occurred during both exponential phase and nitrogen-limited stationary phase. However, the decreased cell yield and higher oxygen consumption of SDS-grown cells were mimicked by cells grown in equivalent concentrations of sucrose or polyethylene glycol. We attribute part 2 of the energy dependence to SDS as a solute. Finally, with regard to the as yet unidentified bacterial osmotic stress detector, we used the micelle-forming nature of SDS to conclude that the detector was responding to turgor pressure-water activity rather than to osmolarity itself.

Lipoxygenase inhibitors shift the yeast/mycelium dimorphism in Ceratocystis ulmi.

The yeast-mycelium dimorphism in Ceratocystis ulmi, the causative agent of Dutch elm disease, was switched by gossypol, nordihydroguaiaretic acid, and propylgallate. In each case the mycelial form was converted to the yeast form. These compounds are recognized lipoxygenase inhibitors. Inhibitors of cyclooxygenase and thromboxane synthetase did not cause mycelia to shift to the yeast form. We suggest the following two-part hypothesis: (i) that lipoxygenase is a target for antifungal antibiotics and (ii) that many phytoalexins (antimicrobial compounds of plant origin) are targeted toward fungal lipoxygenases. In addition, in a study to determine potential lipoxygenase substrates, a fatty acid analysis indicated that C. ulmi conidiospores contained high levels of oleic, linoleic, and linolenic acids but no arachidonic acid.

Physiology of sporeforming bacteria associated with insects: minimal nutritional requirements for growth, sporulation, and parasporal crystal formation of Bacillus thuringiensis.

A defined medium is described in which 18 strains of Bacillus thuringiensis representing the 12 established serotypes grow, sporulate, and produce a parasporal crystal. This minimal medium contains glucose and salts supplemented with either aspartate, glutamate, or citrate. These organic acids are required and cannot be replaced by vitamin mixtures or succinate even though succinate is taken up at a rate similar to that of aspartate, glutamate, and citrate.

Control of pantothenate accumulation in Agrobacterium tumefaciens.

Two pantothenate-requiring mutants of Agrobacterium tumefaciens have been isolated. One of them (strain WMP-1) is unusual in that growth levels equivalent to the parent strain are achieved only when the medium is additionally supplemented with aspartate or another compound related to the tricarboxylic acid cycle. Extracts of cells grown on limiting aspartate were found to contain four times more (14)C-pantothenate than those grown at optimal aspartate concentrations. This difference was found in both the perchloric acid-soluble and -insoluble fractions, presumably the coenzyme A pool and acyl carrier protein, respectively. These findings are discussed in terms of membrane integrity and the control of fatty acid biosynthesis.