Pseudomonas aeruginosa is able to tolerate exposure to high concentrations of β-lactam antibiotics. We have found that when treated with β-lactams, P. aeruginosa undergoes a rapid en masse transition from normal rod shaped cells to viable, cell wall defective (CWD) spherical cells. Interestingly, we found that when the antibiotic is removed, the entire population of spherical cells quickly converts back to bacillary cells. Our results suggest that these rapid and reversible population-wide morphotype transitions function as a strategy to survive exposure to beta-lactam antibiotics.
We hypothesised that β-lactam-induced CWDs are in fact L-forms and therefore have the capability to proliferate. Indeed, we have found that in the presence of β-lactam antibiotic that CWD cells are able to continue biological metabolism producing more DNA and membrane lipids over time. This suggests that CWDs are capable of proliferation. Time-lapse conventional microscopy and super-resolution OMX 3D-structured illumination microscopy (3D-SIM) demonstrated the capability of CWD cells to proliferate by a number of different mechanisms. Using conventional phase-contrast microscopy, CWD can be seen showing mitosis events resulting in the formation of multiple daughter cells. Additionally, CWD cells showed the ability to form tube-like structures that eventually resolved into several small bodies. CWD cells were also capable of forming extracellular buds as reproductive elements. Interestingly, CWD were also observed to form intracellular buds. We found that the formation of internal buds was the most common proliferation mechanism used by P. aeruginosa CWD cells. Consequently, our results have revealed that the CWD morphotype of P. aeruginosa proliferates via processes which are highly similar to those described in the literature for L-form proliferation of different bacterial species. Interestingly, our studies have shown that the β-lactam-induced CWD of P. aeruginosa have the capability of using a number of alternative proliferation mechanisms.