The effect of levofloxacin concentration on the development and maintenance of antibiotic-resistant clones of Escherichia coli in chemostat culture.

A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg x l(-1)) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h(-1)). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h(-1)). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h(-1)). At levofloxacin concentrations less than 0.03 mg x l(-1), the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin.

The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture.

A recombinant strain of Saccharomyces cerevisiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture. A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms. A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics. In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid. Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid. The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones. Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure. Alterations in plasmid retention characteristics could be associated with host or plasmid. The most stable clone isolated exhibited a approximately 30% improvement of its overall stability (sigma(N+)) and an 80% improvement in productivity, when compared to the parental strain CGpLG. This improved stability was associated with alterations in the plasmid genome.

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae.

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture.

A recombinant strain of Saccharomyces cerevisiae, containing a 2-microns-fragment-based plasmid (pYE alpha a4) was grown under non-selective conditions in continuous culture. The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions. The difference in growth rate (delta mu) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique. Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures. Plasmid loss was delta mu-dominated in cultures grown on defined media whereas delta mu and R were co-dominant in cultures grown on complex medium. Loss rates increased with increasing dilution rate in complex glucose-limited cultures. The reverse was found in defined glucose-limited cultures. Plasmid retention and loss kinetic determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures. Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased delta mu indicated an alternative physiological effect of phosphate limitation on plasmid stability.

Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep-sea deposit feeder.

In 1992, two independent reports based on small-subunit rRNA gene (SSU rDNA) cloning revealed the presence of novel Archaea among marine bacterioplankton. Here, we report the presence of further novel Archaea SSU rDNA sequences recovered from the midgut contents of a deep-sea marine holothurian. Phylogenetic analyses show that these abyssal Archaea are a paraphyletic component of a highly divergent clade that also includes some planktonic sequences. Our data confirm that this clade is a deep-branching lineage in the tree of life.

Plasmid instability in an industrial strain of Bacillus subtilis grown in chemostat culture.

A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d mu) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d mu dominated. Changes in medium composition and the nature of growth limitation caused variations in both d mu and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex medium. R and d mu were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT alpha amylase gene and was associated with increased values of R and d mu. Magnesium limitation in minimal medium caused a significant increase in d mu and a decrease in R.

The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture.

A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.

Ethylene production by soil microorganisms.

Ethylene-producing strains of Penicillium cyclopium and P. crustosum were isolated from soil. These isolates produced ethylene on a variety of carbon growth substrates including phenolic acids. The quantities of ethylene produced on the various substrates varied, and the subtrate-ethylene prosuction pattern for P. cyclopium strains differed significantly from that of P. crustosum strains.

Cold osmotic shock in Saccharomyces cerevisiae.

Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Exponentially growing cells from batch cultures grown in defined medium at 30 C, after being suspended in 0.8 m mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mm MgCl(2), accumulated the nonmetabolizable solutes d-glucosamine-hydrochloride and 2-aminoisobutyrate at slower rates than unshocked cells; shocked cells retained their viability. Storage of unshocked batch-grown cells in buffer at 10 C led to an increase in ability to accumulate glucosamine, and further experiments were confined to cells grown in a chemostat under conditions of glucose limitation, thereby obviating the need for storing cells before use. A study was made of the effect of the different stages in the cold osmotic shock procedure, including the osmotic stress, the chelating agent, and the cold Mg(2+)-containing diluent, on viability and solute-accumulating ability. Growth of shocked cells in defined medium resembled that of unshocked cells; however, in malt extract-yeast extract-glucose-peptone medium, the shocked cells had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low-molecular-weight compounds and about 6 to 8% of the cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase and l-leucine-beta-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase, were released when yeast cells were subjected to cold osmotic shock.