Transcriptional stress response in foodborne pathogens

Aaron M Pleitner, Purdue University

Abstract

Novel molecular method use is leading to better identification and understanding of microbial presence in food and food processing environments. The application of molecular methods targeting foodborne pathogens provides genetic profiling and targeted intervention methods. Whole-genome sequencing (WGS) provides information on the genomic uniqueness of specific pathogens and is being implemented in epidemiological investigations. Transcriptomic profiling allows for tracking the differential expression of genes and elucidating stress responses. Effective methods and treatments require an understanding of intervention (e.g. sanitizers) and treatment (e.g. antibiotics) mechanisms and efficacies. Accurate novel molecular method use requires proper learning of theory and data management. Chlorine dioxide (ClO2) is a popular sanitizer in the food industry, yet the mechanism of bacterial inactivation has not been fully elucidated. Here, we examined the response of Listeria monocytogenes to sublethal concentrations of aqueous ClO2 was assessed. L. monocytogenes was grown to early log phase in BHI broth and exposed to ClO2 concentrations of 100, 300, and 500mg/L; assessment identified a concentration of 500mg/L to be significantly differently than 100 and 300 mg/L (p-value<0.05). Transcriptional profiling of L. monocytogenes exposed to 300mg/L ClO2 was performed by microarray with select genes confirmed by qRT-PCR. Microarray analysis identified 340 genes as differentially expressed with 223 genes upregulated (fold change ≥1.5; adj. p-value <0.05). Analysis revealed 113 and 16 genes differentially expressed belonging to major stress regulons σB and CtsR, respectively. Phenotypic screening with isogenic ΔsigB and ΔctsR mutants revealed the absence of these regulators increased susceptibility to ClO2. This study elucidates the bacterial response to a popular sanitizer and provides evidence to effectively combine ClO2 with other intervention methods. Infections related to methicillin-resistant Staphylococcus aureus (MRSA) first originated in the hospital setting. MRSA is now acquired in community settings, in the general population consisting of patients with no recent hospital exposure. MRSA has been isolated during screening studies of livestock yards and food products along the food continuum. Transcriptome analysis of typically applied stressors provides an understanding of resistance mechanisms responsible for bacterial persistence. RNA-Sequencing was used to assess the global transcriptome of MRSA during normal growth conditions (early log and stationary phase) and typical stressors of osmotic and antibiotic pressures. The response to bacteriostatic antibiotics and the role of the major stress regulon, σB, during osmotic stress was elucidated. The activity of σB, encoded by rpoF, was investigated during osmotic stress (NaCl) applied to MRSA JE2 wildtype and JE2 rpoF::TnT, revealing 371 differentially expressed genes (adj. p-value <0.05). The virulence regulators agr and sar were upregulated in the rpoF::TnT strain. Three antibiotic resistance genes were downregulated. Exposure to tetracycline and vancomycin resulted in 1641 and 311 differentially expressed genes (adj. p-value <0.05), respectively. Downregulated genes encoding for metabolic functions and cell division were overrepresented for both antibiotics. While the implementation of new molecular methods is occurring, updated course materials are essential in training young Food Safety professionals. Lecture- and laboratory-based modules on PCR and PFGE were introduced into an existing undergraduate Food Microbiology course for at least 3 semesters. The material was developed to engage undergraduates with interactive group activities, hands-on analysis of data, and group discussions. Normalized change analysis of pre- and post-instruction evaluations revealed increases in student learning of (0.60 ± 0.32 and 0.50 ± 0.33) for PCR lecture and lab and (0.51 ± 0.30 and 0.56 ± 0.38) for PFGE lecture and lab. Effects of academic year were observed, suggesting that material may need to be adjusted to fit student aptitude. A Case-study based course was developed and taught to higher level undergraduates and graduate students at four separate universities. All students had a background in Food Safety. Four case studies were developed and involved molecular and epidemiological methods typically used in outbreak investigations. Evaluations revealed significant increases in learnings with overall mean normalized change increase of 0.32 ± 0.35 between pre- and post-instruction scores. Comparison of individual and group learnings revealed that institution, instruction, learning environment and pre-evaluation scores played a role in overall learnings. Both studies provide platforms for introducing the theory and use of molecular methods. The application of molecular methods allowed for the elucidation of foodborne pathogen stress response to popular intervention strategies. The mechanism of ClO2 was investigated by observing the transcriptional response of L. monocytogenes. The σB regulon of MRSA was defined by RNA-Seq. The transcriptional responses of MRSA to sublethal concentrations of antibiotics, tetracycline and vancomycin, were also tracked. Effective teaching methods involving molecular method theory and use are presented here. Adequate molecular method use is pivotal to pursue a safer food supply and properly investigate outbreak investigations. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Oliver, Purdue University.

Subject Area

Molecular biology|Food Science|Pedagogy

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