Influence of infected cell growth state on bacteriophage reactivation levels.

Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1. 5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.

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.

Microbiology of the oil fly, Helaeomyia petrolei.

Helaeomyia petrolei larvae isolated from the asphalt seeps of Rancho La Brea in Los Angeles, Calif., were examined for microbial gut contents. Standard counts on Luria-Bertani, MacConkey, and blood agar plates indicated ca. 2 x 10(5) heterotrophic bacteria per larva. The culturable bacteria represented 15 to 20% of the total population as determined by acridine orange staining. The gut itself contained large amounts of the oil, had no observable ceca, and maintained a slightly acidic pH of 6.3 to 6.5. Despite the ingestion of large amounts of potentially toxic asphalt by the larvae, their guts sustained the growth of 100 to 1,000 times more bacteria than did free oil. All of the bacteria isolated were nonsporeformers and gram negative. Fourteen isolates were chosen based on representative colony morphologies and were identified by using the Enterotube II and API 20E systems and fatty acid analysis. Of the 14 isolates, 9 were identified as Providencia rettgeri and 3 were likely Acinetobacter isolates. No evidence was found that the isolates grew on or derived nutrients from the asphalt itself or that they played an essential role in insect development. Regardless, any bacteria found in the oil fly larval gut are likely to exhibit pronounced solvent tolerance and may be a future source of industrially useful, solvent-tolerant enzymes.