Quaternary ammonium compounds such as benzalkonium chloride (BC) are widely used as disinfectants in both food processing and medical environments. BC-resistant strains of Listeria monocytogenes have been implicated in multistate outbreaks of listeriosis and have been frequently isolated from food processing plants. However, the genetic basis for BC resistance in L. monocytogenes remains poorly understood. In this study, we have characterized a plasmid (pLM80)-associated BC resistance cassette in L. monocytogenes H7550, a strain implicated in the 1998-1999 multistate outbreak involving contaminated hot dogs. The BC resistance cassette (bcrABC) restored resistance to BC (MIC, 40 g/ml) in a plasmid-cured derivative of H7550. All three genes of the cassette were essential for imparting BC resistance. The transcription of H7550 BC resistance genes was increased under sublethal (10 g/ml) BC exposure and was higher at reduced temperatures (4, 8, or 25°C) than at 37°C. The level of transcription was higher at 10 g/ml than at 20 or 40 g/ml. In silico analysis suggested that the BC resistance cassette was harbored by an IS1216 composite transposon along with other genes whose functions are yet to be determined. The findings from this study will further our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to the elucidation of possible BC resistance dissemination in L. monocytogenes.Listeria monocytogenes is a food-borne pathogen associated with severe illness (listeriosis) in at-risk individuals, including those in extremes of age, pregnant women and their fetuses, and those with compromised immunity. Environmental contamination with this pathogen plays a key role in the eventual contamination of ready-to-eat foods and subsequent foodborne illness (16,19,30). Biofilm formation and persistence, resistance to disinfectants, resistance to Listeria-specific viruses, and the ability to replicate at low temperatures are among the attributes contributing to the organism's prevalence and persistence in food processing environments (7,18,19).Resistance to quaternary ammonium disinfectants such as benzalkonium chloride (BC) is especially relevant to Listeria's adaptations in food-related environments, as these compounds are used extensively in food processing, in retail, and for household or personal use (24,26). BC resistance of L. monocytogenes isolated from foods and from the processing plant environment has been found to range from 10% (1) to as much as 42 to 46% (25, 27, 37). A study of strains from turkey processing plants revealed that resistance to BC was especially high among those of serotype 1/2a (or 3a) and 1/2b (or 3b) (60% and 51%, respectively) and that all BC-resistant strains were also resistant to the heavy metal cadmium (27).Mechanisms underlying BC resistance in L. monocytogenes remain poorly understood. Several studies have provided evidence for chromosomal determinants (6,34,35,37,38), and evidence for plasmid-mediated resistance to BC also exists (21,34,35), ev...
Listeria monocytogenes is a leading agent for severe food-borne illness and death in the United States and other nations. Even though drug resistance has not yet threatened therapeutic interventions for listeriosis, selective pressure associated with exposure to antibiotics and disinfectants may result in reduced susceptibility to these agents. In this study, selection of several L. monocytogenes strains on either ciprofloxacin (2 g/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 g/ml) led to derivatives with increased MICs not only to these agents but also to several other toxic compounds, including gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenylphosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC. Inclusion of strains harboring the large plasmid pLM80 revealed that MICs to ciprofloxacin and gentamicin did not differ between the parental and plasmid-cured strains. However, ciprofloxacin-selected derivatives of pLM80-harboring strains had higher MICs than those derived from the plasmid-cured strains. Susceptibility to the antimicrobials was partially restored in the presence of the potent efflux inhibitor reserpine. Taken together, these data suggest that mutations in efflux systems are responsible for the multidrug resistance phenotype of strains selected on ciprofloxacin or BC.
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 ...
Listeria monocytogenes epidemic clone II (ECII) strains are unusual in being completely resistant to phage when grown at low temperatures (<30°C). In the current study we constructed and characterized a mariner-based mutant (J46C) of the ECII strain H7550-Cd S that lacked temperature-dependent resistance to phage. The transposon was localized in LMOh7858_2753 (open reading frame [ORF] 2753), a member of a 12-ORF genomic island unique to ECII strains. ORF 2753 and ORF 2754 exhibited homologies to restriction endonucleases and methyltransferases associated with type II restriction-modification (RM) systems. In silico-based predictions of the recognition site for this putative RM system were supported by resistance of DNA from ECII strains to digestion by BfuI, a type II restriction enzyme specific for GTATCC (N6/5). Similarly to J46C, a mutant harboring an in-frame deletion of ORF 2753 was susceptible to phage regardless of temperature of growth (25°C or 37°C). Genetic complementation restored phage resistance in 25°C-grown cells of ORF 2753 mutants. Reverse transcription (RT) and quantitative realtime PCR data suggested enhanced transcription of ORF 2753 at low temperatures (<25°C) compared to 37°C. In contrast, available transcriptional data suggested that the putative methyltransferase (ORF 2754) was constitutively expressed at all tested temperatures (4 to 37°C). Thus, temperature-dependent resistance of L. monocytogenes ECII to phage is mediated by temperature-dependent expression of the restriction endonuclease associated with a novel RM system (LmoH7) unique to this epidemic clone.L isteria monocytogenes remains a major food-borne pathogen for at-risk populations, including pregnant women and their fetuses, the elderly, and patients with compromised immunity. Even though listeriosis is relatively infrequent, it is accompanied with high mortality and severe symptoms, such as septicemia, meningitis, and stillbirths (38,45).A number of genotypic tools have revealed that strains from different outbreaks can be closely related, constituting epidemic clones (6,8,9,10,22,42,50). Epidemic clone I (ECI) and epidemic clone II (ECII) have been most extensively characterized. ECI strains have been responsible for numerous outbreaks of listeriosis in North America and Europe, with the first documented outbreak being the coleslaw-associated outbreak in the Maritime Provinces, Canada (8, 9, 22, 42). In contrast, ECII was not recognized until the 1998-1999 multistate outbreak of listeriosis in the United States, which was attributed to contaminated hot dogs. ECII was subsequently implicated in another multistate outbreak in 2002 that involved contaminated turkey deli meats, as well as in an outbreak in Belgium (4, 5, 23, 52; M. Yde, personal communication).A special phenotypic characteristic of ECII strains is their temperature-dependent resistance to phage. In contrast to all other screened Listeria strains, ECII strains failed to form plaques when the bacteria were grown at temperatures below 30°C but did so following gr...
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