Combatting cyanobacteria with hydrogen peroxide: a laboratory study on the consequences for phytoplankton community and diversity.

Experiments with different phytoplankton densities in lake samples showed that a high biomass increases the rate of hydrogen peroxide (HP) degradation and decreases the effectiveness of HP in the selective suppression of dominant cyanobacteria. However, selective application of HP requires usage of low doses only, accordingly this defines the limits for use in lake mitigation. To acquire insight into the impact of HP on other phytoplankton species, we have followed the succession of three phytoplankton groups in lake samples that were treated with different concentrations of HP using a taxa-specific fluorescence emission test. This fast assay reports relatively well on coarse changes in the phytoplankton community; the measured data and the counts from microscopical analysis of the phytoplankton matched quite well. The test was used to pursue HP application in a Planktothrix agardhii-dominated lake sample and displayed a promising shift in the phytoplankton community in only a few weeks. From a low-diversity community, a change to a status with a significantly higher diversity and increased abundance of eukaryotic phytoplankton species was established. Experiments in which treated samples were re-inoculated with original P. agardhii-rich lake water demonstrated prolonged suppression of cyanobacteria, and displayed a remarkable stability of the newly developed post-HP treatment state of the phytoplankton community.

Simulation of the rate of transfer of antibiotic resistance between Escherichia coli strains cultured under well controlled environmental conditions.

It was demonstrated that the tetracycline resistance plasmid in Escherichia coli resembling K-12 23:06 containing the E. coli plasmid DM0133 could be transferred to tetracycline sensitive E. coli K-12 MG1655 YFP. The sensitive recipient strain has a slight metabolic advantage in continuous fermentation in absence of tetracycline pressure and as a result the numbers of the resistant recipient strain increase during fermentation. In presence of tetracycline pressure the sensitive strain is eliminated, but when it acquires tetracycline resistance the strain has still the same metabolic advantage as its sensitive parent strain in absence of tetracycline. Here a model will be shown that could explain the rate of transformation of a sensitive into a resistant recipient strain and its subsequent growth during continuous fermentation. According to the model the probability of formation of mutants would be much higher at the dilution rate of 0.09 compared to 0.28, whereas the growth of mutants would be much faster at high dilution rate. The growth model shows how the recipient mutants and the donor cells behave in relation to the dilution rate and the number of mutants. Apart from a deterministic model describing the growth rate of both the donor strain and the resistant recipient strain a stochastic model was developed that is particularly useful when low numbers of mutants are formed.

Interaction between mutations and regulation of gene expression during development of de novo antibiotic resistance.

Bacteria can become resistant not only by horizontal gene transfer or other forms of exchange of genetic information but also by de novo by adaptation at the gene expression level and through DNA mutations. The interrelationship between changes in gene expression and DNA mutations during acquisition of resistance is not well documented. In addition, it is not known whether the DNA mutations leading to resistance always occur in the same order and whether the final result is always identical. The expression of >4,000 genes in Escherichia coli was compared upon adaptation to amoxicillin, tetracycline, and enrofloxacin. During adaptation, known resistance genes were sequenced for mutations that cause resistance. The order of mutations varied within two sets of strains adapted in parallel to amoxicillin and enrofloxacin, respectively, whereas the buildup of resistance was very similar. No specific mutations were related to the rather modest increase in tetracycline resistance. Ribosome-sensed induction and efflux pump activation initially protected the cell through induction of expression and allowed it to survive low levels of antibiotics. Subsequently, mutations were promoted by the stress-induced SOS response that stimulated modulation of genetic instability, and these mutations resulted in resistance to even higher antibiotic concentrations. The initial adaptation at the expression level enabled a subsequent trial and error search for the optimal mutations. The quantitative adjustment of cellular processes at different levels accelerated the acquisition of antibiotic resistance.

Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains.

Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most.

De novo acquisition of resistance to three antibiotics by Escherichia coli.

The acquisition of resistance to amoxicillin, tetracycline, and enrofloxacin by Escherichia coli MG 1655 was examined by exposing growing cells to constant or stepwise increasing concentrations of these compounds. The minimal inhibitory concentration (MIC) of E. coli for amoxicillin increased from 4-8 to 32 µg/ml after growth in the presence of 1.25 or 2.5 µg/ml. By stepwise increasing the exposure, an MIC of 512 µg/ml was reached. This high MIC was maintained after removal of the antibiotics, whereas the lesser increase after exposure to low levels was reversed, indicating that the high MIC was due to a genetic change, but the lower one to phenotypic adaptation only. The MIC for tetracycline increased from 2 µg/ml to maximally 32 µg/ml. The MIC decreased to control levels in the absence of tetracycline, so no genetic changes seem to have occurred. The MIC for enrofloxacin increased from 0.25 µg/ml to maximally 512 µg/ml depending on the concentration during growth. These data mostly support the "radical-based" theory that bactericidal antibiotics induce a common mechanism that contributes to cell killing. Our findings indicate that exposure to low levels of antibiotics causes an increase in MIC above the concentration that the cells were exposed to. The implication is that exposure to low levels of antibiotics should be prevented as much as possible, because this causes resistance far more than high concentrations that inhibit growth or kill the cell and thus prevent acquisition of resistance.