Phenotypic and genetic characterization of Listeria monocytogenes from the environment of turkey processing plants

Abstract

Resistance of clinical strains of Listeria monocytogenes to heavy metals, especially cadmium and arsenic has been used extensively for subtyping. Furthermore, the three most recent multistate outbreaks of listeriosis in the United States (1998-99 hot dog outbreak, and outbreaks in 2001 and 2002) have involved cadmium resistant strains two of which (1998-99 and 2001) were resistant to the quaternary ammonium disinfectant benzalkonium chloride (BC) as well. In these outbreak strains, genes mediating cadmium and BC resistance were found to be localized in two different gene cassettes on large plasmids (ca. 80 kb). However, limited information has been available on resistance to cadmium, arsenic and BC among L. monocytogenes isolates from the food processing plant environment. It is not known whether resistance to heavy metals (cadmium and arsenic) and BC is plasmid-borne in such strains and whether resistance to cadmium and arsenic may be correlated to resistance to disinfectants commonly used in the processing plant environment. Furthermore, information regarding contamination patterns of turkey processing plants in the United States with regards to different serotypes and strain types of L. monocytogenes is currently inadequate. In this study, we characterized 123 L. monocytogenes isolates (53 of serotype 1/2a or 3a, 39 of serotype 1/2b or 3b, and 27 of the serotype 4b complex, consisting of serotype 4b and the closely related serotypes 4d and 4e, and 4 strains of serotype 1/2c or 3c) from the environment of six turkey processing plants in the United States for cadmium, arsenic and BC resistance. In addition, these isolates were subtyped using pulsed field gel electrophoresis (PFGE). Moreover we investigated the prevalence among these isolates of three different cadmium resistance cadA determinants identified to date in L. monocytogenes: cadA1 (associated with Tn5422), cadA2 (associated with plasmid pLM80, identified in the 1998-99 outbreak strain), and cadA3, associated with the integrated conjugative element (ICE) of L. monocytogenes EGD-e, respectively). We employed plasmid curing protocols to examine if the resistance to heavy metals (cadmium and arsenic) and to BC was plasmid-borne and to assess the stability of cadmium and BC resistance among isolates of L. monocytogenes from the processing plant environment. Resistance to cadmium and BC was more common in serogroup 1/2 strains than 4b, whereas arsenic resistance was more commonly encountered in strains from the serotype 4b complex. We found substantial strain diversity in the turkey processing plants with 104 distinct PFGE types from 123 isolates using both enzymes (AscI and ApaI), resulting in Simpson’s index of diversity (D) of 0.995. Plant-specific strains were commonly found within each serotype. Heavy metal (cadmium and arsenic) and BC resistance were detected frequently among the serogroup 1 /2 strains. Our investigation has shown that 28% of the L. monocytogenes strains harbored both cadA1 and cadA2 and that the type of determinant (cadA1 vs. cadA2) may vary based on serotype as well as based on BC resistance (cadA2 being found more frequently among BC resistant than BC susceptible strains of the same serotype). The results also showed that the curing of plasmid borne cadmium and BC resistance was possible in certain strains isolated from the environment of turkey processing plants although the rate of plasmid loss was low. These results suggest that the processing plant environment may represent a reservoir for L. monocytogenes having resistance to cadmium and BC. Further studies are needed to elucidate the mechanisms underlying the prevalence of the different determinants, and their possible role in the ecology and evolution of L. monocytogenes in the processing plant environment. In addition, further studies need to be undertaken to confirm the location of the resistance determinants on plasmids, or possibly on the chromosome, especially in strains that failed to lose resistance following exposure to plasmid curing protocols.