Potential of oxygen and nitrogen reactive intermediates to disperse Listeria monocytogenes from biofilms

Abstract

Studying biofilm dispersal is important to prevent Listeria monocytogenes persistence in food processing plants and to avoid finished product contamination. Reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) may trigger cell detachment from many bacterial species biofilms, but their roles in L. monocytogenes biofilms have not been fully investigated. This study reports on ROI and RNI quantification in Listeria monocytogenes biofilms formed on stainless steel and glass surfaces; bacterial culture and microscopy combined with fluorescent staining were employed. Nitric oxide (NO) donor and inhibitor putative effects on L. monocytogenes dispersal from biofilms were evaluated, and transcription of genes (prfA, lmo 0990, lmo 0807, and lmo1485) involved in ROI and RNI stress responses were quantified by real-time PCR (qPCR). Microscopy detected the reactive intermediates NO, peroxynitrite, H₂O₂, and superoxide in L. monocytogenes biofilms. Neither NO donor nor inhibitors interfered in L. monocytogenes growth and gene expression, except for lmo0990, which was downregulated. In conclusion, ROI and RNI did not exert dispersive effects on L. monocytogenes biofilms, indicating that this pathogen has a tight control for protection against oxidative and nitrosative stresses.

Keywords: Dispersal; RNI; ROI; Reactive nitrogen intermediate; Reactive oxygen intermediate.

Fig. 1

a–h Confocal laser scanning micrographs of 8-day-old biofilms of L. monocytogenes IAL633 and L. monocytogenes ATCC19115 grown on glass chamber—system B. Images were acquired with a Leica TCS SP5-AOBS microscope, equipped with argon (488 nm) and helium (543 nm) lasers. Results were documented with the software Leica LAS AF version 2.6.0 build 7266. Bright light micrographs were included to show that bacterial cells were present in the fields selected, regardless of specific stainings. Staining for hydrogen peroxide was done with H₂DCF (red fluorescence would indicate positive results); nitric oxide was detected with DAFFM-DA (green fluorescence would indicate positive results); staining for superoxide radicals was done with HEt (red fluorescence would indicate positive results); and peroxynitrite was detected with DHR (red fluorescence would indicate positive results). Scale bar = 10 μm

Fig. 2

Relative gene transcription levels normalized with reference to 16S rRNA (arbitrary units) for free-floating L. monocytogenes cells detached from biofilms (system A—stainless steel coupon, brain heart infusion broth, 8 days of incubation, 25 °C). Treatments were done for 1 h at 25 °C: NO donor (SNP at 2000 μg ml⁻¹) or NO inhibitors (L-NAME and c-PTIO, each one at 100 μM). Dotted lines indicate the expression rate of 1 (no change in comparison with the untreated control) and P < 0.05 was considered for evaluation of statistically significant difference