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...

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 ...

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.

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...

The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated. Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of E. coli slt-II mRNA is useful for detection of viable STEC in ground beef.Escherichia coli O157:H7 was first isolated as a human pathogen in 1982 and has since become a well-known agent of food-borne illness. Cases of hemorrhagic colitis, hemolytic uremic syndrome, and death have been reported following the consumption of raw or undercooked ground beef (8). Although most commonly associated with foods of animal origin, enterohemorrhagic E. coli (EHEC) may also be isolated from contaminated drinking water and acidic foods such as mayonnaise, salad dressings, and buttermilk (6,16,29). The infectious dose for E. coli O157:H7 and other serotypes of EHEC varies greatly between populations. For susceptible individuals such as the elderly and infants, the infectious dose may be as low as 10 cells (8).Without an animal model, there is much controversy over which virulence factors of EHEC contribute to human pathogenicity (8). EHEC produce Shiga-like toxins (SLTs) that characterize this group of pathogenic E. coli. Other virulence markers include intimin, hemolysin, and the locus of enterocyte effacement (8). Food-borne illnesses have occurred with isolates that possess all or only a few of these markers (8, 9). Possession of slt genes is a consistent virulence marker (1), but without evidence of human illness, E. coli possessing slt are often referred to as Shiga toxin-producing E. coli (STEC). Despite the fact that EHEC strains containing slt-I and slt-II have been isolated from patients with hemorrhagic colitis, studies have shown that strains possessing only slt-II are more frequently associated with human disease complications (8, 25).Molecular methods for the detection of E. coli O157:H7 are becoming more widely accepted as an alternative to traditional growth-based tests. The ability to specifically identify the presence of an organism within a fraction of the time required by standard approaches makes molecular tests ideal for use in food systems (2). A number of recent reports have applied molecular tools for specific detection of pathogenic E. coli in food systems (10,24,31,32). Most of these assays have used DNA as the target molecule; however, recent literature has sh...

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Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.