Postadaptational Resistance to Benzalkonium Chloride and Subsequent Physicochemical Modifications of
            <i>Listeria monocytogenes</i>

There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 g/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

Section: Discussionmentioning

confidence: 99%

Characterization of Monolaurin Resistance in Enterococcus faecalis

Dufour¹,

Manson²,

Bremer

et al. 2007

“…Microbial cell surface hydrophobicity (CSH) was determined by the microbial adhesion to solvents (MATS) test based on affinity for nonpolar solvents (20). Xylene was used as the hydrocarbon phase.…”

mentioning

confidence: 99%

Exposure of Escherichia coli ATCC 12806 to Sublethal Concentrations of Food-Grade Biocides Influences Its Ability To Form Biofilm, Resistance to Antimicrobials, and Ultrastructure

Capita

Riesco-Peláez

Alonso-Hernando

et al. 2014

119

b Escherichia coli ATCC 12806 was exposed to increasing subinhibitory concentrations of three biocides widely used in food industry facilities: trisodium phosphate (TSP), sodium nitrite (SNI), and sodium hypochlorite (SHY). The cultures exhibited an acquired tolerance to biocides (especially to SNI and SHY) after exposure to such compounds. E. coli produced biofilms (as observed by confocal laser scanning microscopy) on polystyrene microtiter plates. Previous adaptation to SNI or SHY enhanced the formation of biofilms (with an increase in biovolume and surface coverage) both in the absence and in the presence (MIC/2) of such compounds. TSP reduced the ability of E. coli to produce biofilms. The concentration of suspended cells in the culture broth in contact with the polystyrene surfaces did not influence the biofilm structure. The increase in cell surface hydrophobicity (assessed by a test of microbial adhesion to solvents) after contact with SNI or SHY appeared to be associated with a strong capacity to form biofilms. Cultures exposed to biocides displayed a stable reduced susceptibility to a range of antibiotics (mainly aminoglycosides, cephalosporins, and quinolones) compared with cultures that were not exposed. SNI caused the greatest increase in resistances (14 antibiotics [48.3% of the total tested]) compared with TSP (1 antibiotic [3.4%]) and SHY (3 antibiotics [10.3%]). Adaptation to SHY involved changes in cell morphology (as observed by scanning electron microscopy) and ultrastructure (as observed by transmission electron microscopy) which allowed this bacterium to persist in the presence of severe SHY challenges. The findings of the present study suggest that the use of biocides at subinhibitory concentrations could represent a public health risk.

“…BC resistance has been detected in L. monocytogenes strains of different serotypes and from diverse sources (11)(12)(13)(14). Characterization of L. monocytogenes from foods and food processing plants revealed that most BC-resistant (BC r ) isolates were also resistant to the heavy metal cadmium, although the reverse was not always the case (14).…”

mentioning

confidence: 99%

Conservation and Distribution of the Benzalkonium Chloride Resistance Cassette bcrABC in Listeria monocytogenes

Dutta

Elhanafi

Kathariou

2013

109

bAnalysis of a panel of 116 Listeria monocytogenes strains of diverse serotypes and sources (clinical, environment of food processing plants, and food) revealed that all but one of the 71 benzalkonium chloride-resistant (BC r ) isolates harbored bcrABC, previously identified on a large plasmid (pLM80) of the 1998-1999 hot dog outbreak strain H7858. In contrast, bcrABC was not detected among BC-susceptible (BC s ) isolates. The bcrABC sequences were highly conserved among strains of different serotypes, but variability was noted in sequences flanking bcrABC. The majority of the BC r isolates had either the pLM80-type of organization of the bcrABC region or appeared to harbor bcrABC on the chromosome, adjacent to novel sequences. Transcription of bcrABC was induced by BC (10 g/ml) in strains of different serotypes and diverse bcrABC region organization. These findings reveal widespread dissemination of bcrABC across BC r L. monocytogenes strains regardless of serotype and source, while also suggesting possible mechanisms of bcrABC dissemination across L. monocytogenes genomes. Listeria monocytogenes is a food-borne pathogen that can cause severe illness and death in susceptible individuals (pregnant women and their fetuses, the elderly, and patients with various types of immunosuppression) (1, 2). An array of adaptations, including biofilm formation, cold tolerance, and resistance to disinfectants and to phage, can contribute to the ability of this pathogen to persist in food processing environments and thus contaminate ready-to-eat products (3-8). However, our understanding of the mechanisms and factors affecting such adaptations remains limited.Quaternary ammonium compounds such as benzalkonium chloride (BC) are extensively used in food processing and health care environments (9, 10). BC resistance has been detected in L. monocytogenes strains of different serotypes and from diverse sources (11)(12)(13)(14). Characterization of L. monocytogenes from foods and food processing plants revealed that most BC-resistant (BC r ) isolates were also resistant to the heavy metal cadmium, although the reverse was not always the case (14). Two distinct cadmium resistance determinants (cadA1 and cadA2) were identified among these BC r isolates, alone or together (14, 15). Of these, cadA1 is harbored on Tn5422, associated with plasmids of various sizes (16-18), whereas cadA2 has been identified on large plasmids, such as pLM80 of L. monocytogenes H7858, implicated in the 1998-1999 hot dog-associated outbreak of listeriosis, and pLI100 of L. innocua CLIP 11262 (17,19,20).In pLM80 of L. monocytogenes H7858, cadA2 appears to be a component of a composite transposon flanked by IS1216 elements (IS1216 left and IS1216 center in Fig. 1A). In the vicinity of cadA2 a three-gene cassette (bcrABC) was found to be associated with resistance to quaternary ammonium disinfectants such as BC. This cassette also appears to be a component of two putative transposable units, one flanked by IS1216 center and IS1216 right and the other flanked by ...