Abstract
Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).
Keywords: Persister cells, Pseudomonas aeruginossa, Biofilms, Tube reactors, Planktonic populations
Materials and Reagents
Equipment
Procedure
The procedure for persister cell isolation relies on activation of the SOS response together with stringent response, through the use of ciprofloxacin as an antibiotic of choice (Keren et al., 2004; Sufya et al., 2003; Dörr et al., 2009) and saline as carrier solution (Sufya et al., 2003). The presence of ciprofloxacin activates the SOS DNA damage response via a mechanism that involves both recA and lexA and all non-persister cells are killed, while at the same time, persister cell formation occurs when a specific high or low level of SOS function is expressed (Dörr et al., 2009). The use of saline as a carrier for ciprofloxacin leads to an absence of nutrients and activates the stringent response where activation of relA and spoT results in increased levels of ppGpp that inhibit rRNA synthesis and lead to a decrease of the cell’s metabolism and a higher number of persister cells (Bernier et al., 2013, Potrykus and Cashel, 2008). Following isolation of persister cells from planktonic or biofilm cell populations, the persister cell state must be confirmed by performing the part C.
Representative data
Using this methodology, persister cells are isolated using the SOS response, as a standard procedure (Keren et al., 2004; Dörr et al., 2009; Moker et al., 2010; Keren et al., 2004; Pan et al., 2012; Niepa et al., 2012; Hong et al., 2012). When performing persister cell isolation, either in planktonic or biofilm cultures, a typical biphasic killing curve should be observed, in the presence of ciprofloxacin (Figure 1A). Where in the first 3-6 h an exponential decrease of cell number is observed, followed by a killing plateau where no further decrease of cell viability is observed. Cells present in this plateau are considered to be the persister cell sub-population within a bacterial culture and can be from 0.0001 to 0.1% of the total population. Once persister cells are isolated, confirmation of a persister cell state is determined by exposing the isolated cells to ciprofloxacin (20 mg/L) or saline for further 24 h. If cells are truly in a persister state, then no reduction of cell viability is observed (Figure 1B). Figure 1. Isolation of persister cells from biofilm populations of P. aeruginosa. A. Biphasic curve observed upon exposure of P. aeruginosa biofilms to ciprofloxacin (squares) together with control, biofilms exposed to saline (open circles). B. Confirmation of persister cell state, exposure of persister cells to ciprofloxacin (black triangle) does not result in decrease of cell viability compared to control (inverted triangle). (Copyright© 2014, American Society for Microbiology ( Marques et al., 2014).
Notes
Recipes
Acknowledgments
Persister isolation is a modification of previously published protocols (Keren et al., 2004; Sufya et al., 2003). This work was supported by SUNY structural funds.
References
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