Abstract
Conjugation of plasmids is widespread among bacteria and contributes to the spread of antibiotic resistance. In the natural environment, microorganisms predominantly exist in the form of biofilms or other bioaggregates, where they may be exposed to contaminants such as antibiotics, at subinhibitory concentrations. Bacterial cells in older biofilms have lower growth activity due to oxygen and nutrient limitation in the deeper layers of the biofilms. In batch culture, population growth eventually ceases during the stationary phase. Thus, the steady state of biofilms may resemble stationary growth phase cultures. Our objectives were to study (i) the effect of cell growth phases and (ii) subinhibitory and minimum inhibitory concentrations (MIC) of antibiotics on transconjugant formation in both batch cultures and biofilms. Additionally, (iii) the effect of variable nutrient concentrations on MIC was investigated and (iv) an optimization of RT-PCR method for the detection of traA gene (which encodes pilus biosynthesis) expression was carried out. To study the effect of cell growth phases on transconjugant formation, plate matings were carried out utilizing planktonic cultures grown to exponential or stationary phase of donor and recipient strains. The results showed that transconjugant abundance was the highest (20± 0.08%) when both plasmid donor and recipient cells were grown to the stationary phase. However, the growth phase of the donor did not seem to play a role in biofilms. When donor cells were harvested from either the exponential or stationary phase of growth, and inoculated into 24 h old recipient biofilms, there was no statistically significant difference between transconjugant abundance. A higher percentage of transconjugants was detected in plate matings when the donor was exposed to 0.5× minimum inhibitory concentration of gentamicin and additionally challenged with gentamicin at MIC. In biofilms, transconjugant formation was not enhanced when the donor cells were grown with 0.5× MIC gentamicin, and 0.5× MIC gentamicin was added to the biofilms. A decrease in nutrient concentration was associated with a decrease in the MIC. traA expression, detected using RT-PCR in plasmid donor cells grown to early exponential and late exponential phases did not coincide with an increase in transconjugants.
Highlights
Horizontal transfer of mobile genetic elements is an important mechanism of microbial adaptation to a changing environment (Top and Springael, 2003)
To determine if conjugative pilus biosynthesis is linked to a specific growth phase of donor cells and enhanced transconjugant formation, we developed an room temperature (RT)-polymerase chain reaction (PCR) assay to determine the expression of traA gene
Quantitative analysis (Table 4.1) of transconjugant cells and total cells derived from microscopic images (Figure 4.4 and Figure 4.5) followed by statistical analysis revealed that the transconjugants expressed as a percentage of all cells were the highest when both plasmid donor and recipient cells were harvested from the late stationary phase (Figure 4.5), whereas the lowest percentage of transconjugants was detected when the plasmid donor cells were grown to early exponential phase and the recipient cells were harvested from the late stationary phase (Table 4.1)
Summary
Limited information is available on how cell growth phases, of either donor or recipient cells, affect plasmid transfer in planktonic cell batch cultures and in biofilms (Muela et al, 1994). Limited information is available on how cell growth phases (and associated changes in cell size) affect plasmid transfer in planktonic cell batch cultures and in biofilms. Our objectives for this project were to investigate the effect of cell growth phases (exponential and stationary) of plasmid donor and recipient cells on transconjugant formation using planktonic cells in plate mating experiments and to study the effect of different growth phases of the plasmid donor on transconjugant formation in biofilms. We hypothesized that the traA gene expression in donor cells will be linked to a higher percentage of transconjugants
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