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

Abstract

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 ...

Genetic Characterization of Plasmid-Associated Triphenylmethane Reductase in Listeria monocytogenes

“…Previous data indicated that tmr was part of a 3.8-kb transcript that also included bcrABC (mediating resistance to BC). The amounts of this polycistronic transcript increased in the presence of sublethal amounts of BC (22,23). To determine whether the exposure to CV could also result in enhanced amounts of this transcript, we used reverse transcription-PCR (RT-PCR) to assess the impact of CV treatment on the expression of bcrABC.…”

“…An arrow points to the expected spoVG PCR product of 533 bp. Lanes 6 to 10, RT-PCR of bcrABC using primer p2 (for cDNA) and primers p1 and p2 for PCR (22,23), as follows: lane 6, H7550 in the absence of BC or CV; 7, H7550 exposed to BC (10 g/ml); 8, H7550 exposed to CV (15 g/ml); 9 and 10, H7550 genomic DNA and total RNA, respectively, used as a positive and negative control for RT-PCR. Arrow points to the expected bcrABC PCR product of 1130 bp.…”

“…PCR analyses employed the TaKaRa ExTaq kit (TaKaRa, Madison, WI) and a T1 thermal cycler (Biometra, Gottingen, Germany). Primers p1/p2 and 0067F/0067R were used for bcrABC and for tmr, respectively (22,23).…”

“…The cultures were then divided into two equal portions, one of which was supplemented with 15 g/ml CV, while the other was left untreated and incubated at 37°C for 30 min. The total RNA was isolated from the CV-treated and untreated cultures as described previously (22,23,25). RNA was isolated in three independent trials and reverse transcribed as described before (22,23,25).…”

“…RNA was isolated in three independent trials and reverse transcribed as described before (22,23,25). The genespecific cDNA was reverse transcribed using 0067-R for tmr (23) and s2 for the housekeeping gene spoVG, which was used as a reference (22,25,26).…”