The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures

Abstract

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren’t significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes.

Strain and Growth Conditions may Regulate Resistance of Listeria monocytogenes Biofilms to Benzalkonium Chloride

“…Thus, the effect of disinfectants on biofilm formation, by resistant and susceptible strains, can be quantified using RSZ, and RSZ-staining might improve the results obtained with crystal violet staining. For example, with low biofilm-forming prfA mutants [19] and viable but non-culturable (VBNC) cells present in biofilms [29]. Ortiz et al [19] reported that biofilm stimulation by BAC was not observed with BAC-R prfA mutant strains due to the low biofilm formed by prfA mutants, while our unpublished results reveal a better inter-strain reproducibility and sensitivity for RSZ-staining than for crystal violet staining with prfA mutant strains.…”

“…However, a subpopulation of biofilm cells can be VBNC and would not be detected by the CFU approach. It is known that L. monocytogenes and other pathogens are able to survive environmental stresses such as disinfectants by entering into a VBNC state [29]. As there is a good correlation between results obtained with RSZ-based quantification and CFU counts [22], and the lower limit of quantification with the RSZ method can reach 10 3 CFU/biofilm [22], RSZ can detect metabolic activity in VBNC bacteria present in biofilms if the number of cells is above that figure.…”

GtcA is required for LTA glycosylation in Listeria monocytogenes serovar 1/2a and Bacillus subtilis

“…In contrast, absence of the GtrA homolog GtcA results in the loss of sugar modifications on LTA in L. monocytogenes 10403S as well as in B. subtilis 168. The same protein has been previously shown to be important for WTA glycosylation in L. monocytogenes and L. innocua and has been proposed to act as a flippase enzyme ( Promadej et al, 1999 , Lan et al, 2000 , Brauge et al, 2018 ). In addition, a recent study suggested that a GtcA homologue is responsible for the translocation of C 55 -P-GlcNAc during the LTA glycosylation process in S. aureus ( Kho and Meredith, 2018 ).…”

“…Deletion of gtcA in a L. monocytogenes serotype 4b strain has been shown to lead to the loss of sugar modifications on WTA ( Promadej et al, 1999 ). GlcNAc modifications on WTA are important for adhesion, biofilm formation and virulence of L. monocytogenes strains ( Brauge et al, 2018 , Faith et al, 2009 ). While there is evidence in the literature that the absence of GtcA does not impact the structure of LTA in L. monocytogenes serovar 4b strains ( Promadej et al, 1999 ), the recent finding that LTA and WTA are glycosylated by similar mechanisms in L. monocytogenes 10403S, a serovar 1/2a strain ( Rismondo et al, 2018 ) ( Fig.…”

Structure and function of Listeria teichoic acids and their implications

“…Due to the high overall amount of phosphate groups and corresponding electron density, WTAs feature a strong negative charge and confer significant hydrophilicity, contributing to general cation homeostasis (Biswas et al, ), and support biofilm development (Brauge et al, ). This aspect of WTA function could be especially relevant for Listeria , which seems to be specifically adapted to the harsh, ion‐rich and osmotically challenging conditions that can be found in, for example, food production environments (Brauge et al, ). Listeria mutants that lack WTA show severe physiological deficiencies, such as loss of septum positioning, and aberrant morphology (Eugster & Loessner, ).…”