Real-time monitoring of morphological plasticity

Although bacteria have evolved complex molecular strategies to maintain their shape, many are able to alter their shape and size as a survival strategy in response when they encounter stressful environments such as oxidative stress, nutrient limitation, DNA damage and antibiotic exposure. 

With oCelloScope you can now monitor and analyse the bacterial growth and morphological changes simultaneously in standard 96 well plates. Through highly sensitive and automated data acquisition and image analysis, oCelloScope enables scientists to understand the effects and stress reactions of bacteria towards new antibiotic candidates in drug development, as well as on the existing antibiotic tested on putative resistant strains, without additional work load.

 

Below is an example of a full plate AST analyses performed with the BCA algorithm. Morphological changes can quickly be identified using the plate overview provided in our UniExplorer software. In well C5, a bump in the curve can be seen which corresponds to the spheroplast formation and cell burst seen in the video

Acinetobacter boumannii growth analyses captured with oCelloScope: (left) video from C2 – non-treted control, (middle) video from C5 – treated with 0.5µg/ml of Imipenem, (right) UniExplorer plate overview: growth curves automatically calculated with BCA algorithm. Starting cell concentration 105 cell/ml.

Escherichia coli growth time-lapse captured with oCelloScope: (left) non-treted control , (right) treated with 0.12µg/ml of Imipenem: spheroplast formation and cell lysis is observed upon treatment. Starting cell concentration 105 cell/ml.

Spheroplast formation and cell burst

Inhibitors of cell wall synthesis causes growing bacteria to form spheroplasts. A spheroplasts is a cell from which the peptidoglycan component but not the outer membrane component of the cell wall has been removed.

Peptidoglycan synthesis inhibitors such as imipenem, a β-lactam antibiotic, causes the bacteria to swell and form large and fragile spheroplasts when exposed to sub-MIC or MIC concentrations.

Heres an example imipenem induces spheroplasts and cell burst in E. coli captured with oCelloScope

Antibiotic-induced Cell burst_long

β-lactame-induced bacterial filamentation

Filamentation is the anomalous growth of certain bacteria in which cells continue to elongate but do not divide.

Some peptidoglycan synthesis inhibitors, such as ceftazidime, induce filamentation by inhibiting the penicillin binding proteins (PBPs) responsible for crosslinking peptidoglycan at the septal wall (eg. PBP3 in E. coli and P. aeruginosa). 

The special designed SEAL algorithm can detect filamentation of rod-shaped bacteria based on segmentation and extraction of the average bacterial length.

Learn more details and watch the videos in this publication from CDC, McLaughlin and Sue (2018), where SEAL was used to measure the average cell length of B. pseudomallei strains Bp82 and JB039 in the presence and absence of CAZ.

Pseudomonas aeruginosa growth time-lapse captured with oCelloScope: (left) non-treted control, (right) treated with 0.5µg/ml of Ceftazidime: Filamentation is observed as a consequence of antibiotic treatment. Starting cell concentration 105 cell/ml.

Pseudomonas aeruginosa growth time-lapse captured with oCelloScope: (left) non-treted control, (right) treated with 1 µg/ml of Ciprofloxacin: Cell growth inhibition is observed as a consequence of antibiotic treatment. Starting cell concentration 105 cell/ml.

Cell growth inhibition by DNA-replication block

For antibiotics such as fluorochinolones which interfere with the DNA-replication by blocking the DNA-gyrase, the cell growth is completely stopped and the cell death occurs slowly.

In the video an example with P. aeruginosa exposed to the minimum inhibitory concentration of Ciprofloxacin captured with oCelloScope is shown. The cell growth is completely stopped, no observable morphological change occurs and the cells slowly breaks apart.

The possibility of getting fast growth curves combined with morphological dynamics help scientists to understand the effects and stress reactions of bacteria towards antibiotics, without additional work load.

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