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 μg/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 μg/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 μg/ml than at 20 or 40 μg/ml. In silico analysis suggested that the BC resistance cassette was harbored by an IS 1216 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 .

Campylobacter spp., especially Campylobacter jejuni and C. coli , are leading bacterial foodborne pathogens worldwide. In the United States, an estimated 0.8 million cases of campylobacteriosis occur annually, mostly involving C. jejuni . Campylobacteriosis is generally self-limiting, but in severe cases, treatment with antibiotics may be mandated. The increasing incidence of fluoroquinolone resistance in Campylobacter has rendered macrolides such as erythromycin and azithromycin the drugs of choice for human campylobacteriosis. The prevalence of macrolide resistance in C. jejuni remains low, but macrolide resistance can be common in C. coli . Substitutions in the 23S rRNA gene, specifically A2075G, and less frequently A2074C/G, remain the most common mechanism for high-level resistance to macrolides. In C. jejuni , resistance mediated by such substitutions is accompanied by a reduced ability to colonize chickens and other fitness costs, potentially contributing to the low incidence of macrolide resistance. Interestingly, similar fitness impacts have not been noted in C. coli . Also noteworthy is a novel mechanism first reported in 2014 for a C. coli isolate from China and mediated by erm (B) harbored on multidrug resistance genomic islands. The incidence of erm (B) appears to reflect clonal expansion of certain strains, and whole-genome sequencing has been critical to the elucidation of erm (B)-associated macrolide resistance in Campylobacter spp. With the exception of one report from Spain, erm (B)-mediated macrolide resistance has been restricted to Campylobacter spp., mostly C. coli , of animal and human origin from China. If erm (B)-mediated macrolide resistance does not confer fitness costs in C. jejuni , the range of this gene may expand in C. jejuni , threatening to compromise treatment effectiveness for severe campylobacteriosis cases.

Foodborne transmission of Listeria monocytogenes was first demonstrated through the investigation of the 1981 Maritime Provinces outbreak involving coleslaw. In the following two decades, most listeriosis outbreaks involved foods of animal origin, e.g., deli meats, hot dogs, and soft cheeses. L. monocytogenes serotype 4b, especially epidemic clones I, II, and Ia, were frequently implicated in these outbreaks. However, since 2008 several outbreaks have been linked to diverse types of fresh produce: sprouts, celery, cantaloupe, stone fruit, and apples. The 2011 cantaloupe-associated outbreak was one of the deadliest foodborne outbreaks in recent U.S. history. This review discusses produce-related outbreaks of listeriosis with a focus on special trends, unusual findings, and potential paradigm shifts. With the exception of sprouts, implicated produce types were novel, and outbreaks were one-time events. Several involved serotype 1/2a, and in the 2011 cantaloupe-associated outbreak, serotype 1/2b was for the first time conclusively linked to a common-source outbreak of invasive listeriosis. Also in this outbreak, for the first time multiple strains were implicated in a common-source outbreak. In 2014, deployment of whole genome sequencing as part of outbreak investigation validated this technique as a pivotal tool for outbreak detection and speedy resolution. In spite of the unusual attributes of produce-related outbreaks, in all but one of the investigated cases (the possible exception being the coleslaw outbreak) contamination was traced to the same sources as those for outbreaks associated with other vehicles (e.g., deli meats), i.e., the processing environment and equipment. The public health impact of farm-level contamination remains uncharacterized. This review highlights knowledge gaps regarding virulence and other potentially unique attributes of produce outbreak strains, the potential for novel fresh produce items to become unexpectedly implicated in outbreaks, and the key role of good control strategies in the processing environment.

Analysis 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 μg/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.

The resistance of Listeria monocytogenes to cadmium and arsenic has been used extensively for strain subtyping. However, limited information is available on the prevalence of such resistance among isolates from the environment of food-processing plants. In addition, it is not known whether the resistance of such isolates to heavy metals may correlate with resistance to quaternary ammonium compounds extensively used as disinfectants in the food-processing industry. In this study, we characterized 192 L. monocytogenes isolates (123 putative strains) from the environment of turkey-processing plants in the United States for resistance to cadmium and arsenic and to the quaternary ammonium disinfectant benzalkonium chloride (BC). Resistance to cadmium was significantly more prevalent among strains of serotypes 1/2a (or 3a) and 1/2b (or 3b) (83% and 74%, respectively) than among strains of the serotype 4b complex (19%). Resistance to BC was encountered among 60% and 51% of the serotype 1/2a (or 3a) and 1/2b (or 3b) strains, respectively, and among 7% of the strains of the serotype 4b complex. All BC-resistant strains were also resistant to cadmium, although the reverse was not always the case. In contrast, no correlation was found between BC resistance and resistance to arsenic, which overall was low (6%). Our findings suggest that the processing environment of turkey-processing plants may constitute a reservoir for L. monocytogenes harboring resistance to cadmium and to BC and raise the possibility of common genetic elements or mechanisms mediating resistance to quaternary ammonium disinfectants and to cadmium in L. monocytogenes .

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 μg/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 μg/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.