Graduate Thesis Or Dissertation
 

Genomic mapping of efflux pumps in Listeria monocytogenes and their contribution to cadmium and fluoroquinolone tolerance

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  • Listeria monocytogenes is the third most deadly foodborne pathogen in the United States. The young and elderly, as well as pregnant and immunocompromised people are the population most susceptible to serious illness and death from listeriosis infections. Unlike most foodborne pathogens, L. monocytogenes does not live a solely enteric lifestyle. It is naturally found in the environment, including in soil and water. It is a psychrotrophic facultative anaerobe, which gives L. monocytogenes a competitive advantage in refrigerated foods and in food processing environments. Considering these characteristics, it is perhaps not unexpected that L. monocytogenes is commonly found in food processing facilities, occasionally with the same strain persisting for years or even decades. The exact reason for its ability to persist and evade cleaning and sanitizing procedures is likely to be multifaceted. Researchers have theorized that strain persistence may be associated with the possession of efflux pumps: membrane bound proteins that utilize either an ion-gradient or ATP hydrolysis to physically move toxic substances up their concentration gradient and out of the cell. A number of efflux pumps with specificity to a variety of toxic substances have been described in L. monocytogenes, including pumps associated with increased tolerance to heavy metals, antibiotics, and sanitizers. To further our understanding of efflux pumps in L. monocytogenes and how they may contribute to persistence, we focused on studying a set of isolates (n = 88) from British Columbia, Canada. This set of isolates originates from five dairy processing facilities with their isolation spanning over a decade. A majority of the isolates (63/88) were recovered from one facility over a seven-year period that belong to a single clonal group (<16 SNPs in the core genome). Additional L. monocytogenes strains were included for comparison purposes. This set of isolates was previously whole genome sequenced using short-read technology, giving a special opportunity to compare closely related isolates both genotypically and phenotypically. These isolates were challenged with exposure to the toxic heavy metal, cadmium, as well as to a variety of antibiotics with special attention to fluoroquinolones due to efflux pump-mediated resistance mechanisms. The foundational methods of this research were to quantify the response, primarily lag phase duration (LPD), of individual strains to grow in the presence of these toxic compounds. Isolates were challenged against these compounds (cadmium or ciprofloxacin) using a high-throughput 96-well screening assay with Muller-Hinton broth (MHB) at an approximate starting concentration of 5 log CFU/mL. The 96-well plate was placed in a spectrophotometer at 37°C for 24-48 hours with the OD595 read every 10 minutes. Growth curves generated were modeled to ascertain the lag phase duration. Supportive methods included antimicrobial susceptibility testing using the VITEK 2 Compact with the GP-AST75 card as well as Nanopore long-read next-generation sequencing to resolve selected genomes. L. monocytogenes strains with various cadmium efflux pump variants (cadA1-cadA4) were capable of growth in the presence of cadmium salts. ScottA, carrying the cadA4 variant, had the highest MIC at 175 µM of CdCl2. Variants cadA1-cadA3 had similar growth kinetics in the presence of cadmium. The control strain that did not carry cadA (WRLP85) demonstrated an inherent cadmium tolerance of 10.9 µM for L. monocytogenes. All cadA variants were less capable of mediating the toxicity of CdSO4 compared to CdCl2, as demonstrated by significant increases in lag phase duration. This is the first study to demonstrate a difference in L. monocytogenes response to different cadmium salts. When the dairy isolate set (n = 88) was cultured in the presence of 43.8 µM of CdCl2, only strains possessing cadA1 (n = 66) or cadA2 (n = 1) were capable of growth. Within the cadA1 isolate group, 65 of these strains were isolated from a single facility and were very closely related (<33 SNPs), whereas one strain (WRLP95) was not closely related. The impact of cadmium on the lag phase durations of the cadA1 isolates varied significantly, even among the clonal group (one-way ANOVA; p < 0.05). The lag phase duration of WRLP95 was the most negatively impacted by cadmium. All the isolates had identical cadA1C1 sequences; however, WRLP95 carried a different plasmid from the closely related cadA1 group. Plasmids were resolved for selected strains in the closely related cluster in hopes of explaining phenotypic differences; however, all strains, with the exception of WRLP77, carried identical plasmids (0 SNPs) that possessed additional non-specific soft metal efflux genes. Further work in transcriptomics would be needed to ascertain whether differences in expression occur for these closely related strains. All of the L. monocytogenes isolates were susceptible to the tested fluoroquinolones (MIC ciprofloxacin ≤1 ppm, levofloxacin ≤2 ppm, moxifloxacin, ≤1 ppm). However, the strains differed in their ability to grow in the presence of low concentrations (≤ 2 ppm) of these antibiotics. There was a dichotomy of responses to ciprofloxacin, with 47 isolates having a MIC of 1 ppm while 41 isolates had a MIC of ≤0.05 ppm, below the concentration tested on the susceptibility card. All 88 isolates were screened for their ability to grow in the presence of 0.5 ppm ciprofloxacin. There was a large variation in growth responses, even within technical replicates of the same isolate. Generally, isolates with a VITEK-assigned MIC of ≤0.05 ppm demonstrated a significantly longer lag time (15.2 h; Welch’s two sample t-test; p < 0.05) compared to isolates with a VITEK-assigned MIC of 1 ppm (4.45 h). These large variations were hypothesized to be due to mutations during culturing. To test this, isolates demonstrating large LPD variations within technical replicates were chosen for a second passage through 0.5 ppm of ciprofloxacin. A decrease in both LPD and variability within technical replicates were measured suggesting that mutations had occurred. Prior research has identified small mutations in the fepR and topoisomerase genes as being effective mechanisms for increasing bacterial tolerance to fluoroquinolones. Sequencing of these ciprofloxacin-passaged strains would be an effective method to confirm the type and location of the mutation. Recent literature on efflux pumps in L. monocytogenes has focused on genotypic characterization with thorough phenotypic assessments being uncommon. The data presented here suggest comprehensive phenotypic assessments should not be overlooked. There were significant differences in phenotypes that were not supported by genomic comparisons. This discordance, especially between such closely related isolates, should be investigated further. Cumulatively, these data represent a one-of-a-kind phenotypic and genotypic assessment that expands our knowledge of the ability of L. monocytogenes to deal with diverse antimicrobial compounds. 
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