Assessment of the bacterial viability of chlorine‐ and quaternary ammonium compounds‐treated Lactobacillus cells via a multi‐method approach

Abstract

Aims: The assessment of the bacterial viability of chlorine-and quaternary ammonium compounds (QACs)-treated Lactobacillus cells by culture-dependent and -independent methods. Methods and Results: Lactobacillus isolates (Lactobacillus plantarum G1, Lactobacillus plantarum B1, Lactobacillus brevis S1 and Lactobacillus paracasei W1) in biofilm and planktonic cell suspensions were treated with chlorine-based (0Á018 and 0Á18%) and QACs-based (0Á2 and 2Á0%) disinfectants for 5 min and then analysed by plate counting, flow cytometry (FCM) and fluorescence activated cell sorting (FACS). The reaction of sessile cells to disinfectants was assessed with the confocal laser scanning microscopy (CLSM). Plate counts revealed L. paracasei W1 to be substantially inactivated by both disinfectants, while counts of the other isolates to be significantly reduced only by QACs, with L. plantarum B1 and L. brevis S1 showing a greater difference between QACs concentrations and cell types. In several cases, the disinfectants caused slightly higher inactivation of planktonic than biofilm cells, with L. plantarum B1 being significantly less sensitive to QACs in biofilm cells (P < 0Á05). Following FCM with a Syto â 9/PI assay, which addresses cell membrane integrity, the emergence of damaged (Syto â 9 À PI + ) and injured (Syto â 9 + PI + ) subpopulations was often observed in cells when they were treated with QACs, whereas intact (Syto â 9 + PI À ) and unstained (Syto â 9 À PI À ) subpopulations were mostly encountered in chlorine-treated cells. Except Syto â 9 À PI + , all subpopulations were recovered on agar plates following FACS, with biofilm cells showing higher culturability irrespective of conditions, probably because of the residues of the biofilm matrix which serve as a protective cover for the bacteria. The CLSM revealed a substantial cell membrane damage within the QACs-treated biofilms, however, some cells deep in the biofilm were still intact and thus remained protected against this disinfectant. Conclusion: We found that FCM/FACS proved useful in the analysis of lactobacilli membrane integrity in disinfection experiments as well as in recovery evaluation of planktonic-biofilm cell subpopulations. In turn, CLSM was particularly useful in investigating the resistance mechanism when Lactobacillus cells were embedded in biofilms. Significance and Impact of the Study: This study highlights the need for treatment optimization on a case-by-case basis to avoid the emergence of cells in intermediate states with recovery potential and to reach and, thus, kill all bacteria in already developed lactobacilli biofilms.

The Effects of Eugenol, Trans-Cinnamaldehyde, Citronellol, and Terpineol on Escherichia coli Biofilm Control as Assessed by Culture-Dependent and -Independent Methods

“…Previous studies have shown how various food spoilage and pathogenic bacteria can persist due to biofilm formation either on food or in a food production environment, e.g., Escherichia coli O157:H7 [13], Salmonella enteritidis [5], Listeria monocytogenes [14], Pseudomonas aeruginosa [15], Pseudomonas fragi [16], Bacillus cereus [17], and Lactobacillus plantarum [18,19]. It is thus very difficult to control these bacteria within biofilms, despite applying the best antimicrobial agents [20].…”

Biofilm formation by lactobacilli and resistance to stress treatments

“…As cells imposing injury state (Syto ® 9 + PI + ) have already been characterised (Ben‐Amor et al ., ; Berney et al ., ), the phenomenon of Syto ® 9 − PI − cells is not well‐understood. This study show that Syto ® 9 − PI − cells represent an intermediate state with viability reduced to the level dependent on the bacterial cells and conditions applied, which concurs with previous work performed by our group (Olszewska et al ., ). As shown for L. plantarum B1, the ability of Syto ® 9 − PI − cells to grow on agar plates was largely lost by planktonic cells but not by biofilm ones after AA treatment.…”

“…Optical filters were set up so that the red fluorescence was measured above 670 nm (FL4) and the green fluorescence was measured at 520 nm (FL1). Two independent trials of suspended cells were sorted with the same instrument (Beckman Coulter), as described elsewhere (Olszewska et al, 2019).…”

“…Untreated, ET-and AA-treated biofilms were stained with the LIVE/DEAD viability kit (Molecular Probes). An image acquisition was performed using a Zeiss LSM 800 Confocal Laser Scanning Microscope (Carl Zeiss Microscopy, G€ ottingen, Germany) and images taken were processed with COMSTAT (Heydorn et al, 2000), a Matlab-based image analysis software (http://www.imageanalysis.dk/) as described elsewhere (Olszewska et al, 2019).…”

“…In turn, Somers et al (2001) reported that nonstarter lactobacilli formed a biofilm on the equipment used for Cheddar cheese making. The resistance of lactobacillus biofilms is poorly understood too, however, biofilms of L. plantarum were shown to be more resistant to disinfecting agents such as chlorine-based and quaternary ammonium compounds-based than planktonic bacteria (Kubota et al, 2008(Kubota et al, , 2009Olszewska et al, 2019). Studies on lactobacillus biofilms in the context of their use in biotechnological processes are even more scarce.…”