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Title: SYNERGISTIC EFFECT OF FATTY ACIDS AND NISIN IN INHIBITING PERSISTER AND BIOFILM OF LISTERIA MONOCYTOGENES
Creator: Zhou, Jiacheng
Date: 2019
Description: A foodborne pathogen Listeria monocytogenes causes a life-threatening listeriosis in humans after eating contaminated food. The FDA-approved...
Show moreA foodborne pathogen Listeria monocytogenes causes a life-threatening listeriosis in humans after eating contaminated food. The FDA-approved antimicrobial peptide nisin has been used to prevent contamination of food product from Gram-positive pathogens including L. monocytogenes. However, the formation of biofilms and persisters (i.e., metabolically dormant bacterial population) has resulted in the failure of nisin treatment. Fatty acids, which have been known to exhibit antimicrobial activities, are widely used for therapeutics, food preservation, and agriculture. Previously, we found that two fatty acid compounds lauric acids and N-tridecanoic acids are effective in inhibiting biofilms and persister formation of Gram-negative pathogens. In this study, we investigate whether the fatty acid treatment in combination with nisin promotes inactivation of L. monocytogenes, especially biofilms and persisters. The fatty acid-only treatment reduced the level of biofilms and persisters, while nisin-only treatment resulted in the development of resistant population of L. monocytogenes ATCC19115 strain. However, the co-treatment of the fatty acid and nisin synergistically enhanced the killing of L. monocytogenes by significantly decreasing the number of survived cells and inhibiting biofilms. These results are particularly important in improving food safety in that the food-grade fatty acids can be applied to repress the occurrence of resistant mechanisms of foodborne pathogens by inhibiting biofilm and persister cell formation.
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Title: Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator: Raba, Daniel Alexander
Date: 2019
Description: The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
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Title: Role of Respiratory Enzymes on Growth of Pseudomonas aeruginosa in Luria-Bertani and Artificial Urine Media
Creator: Hu, Yuyao
Date: 2018
Description: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes many hospital-acquired infections. The treatment of P. aeruginosa...
Show morePseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes many hospital-acquired infections. The treatment of P. aeruginosa infections is difficult due its multiple defense and adaptive mechanisms. One of these mechanisms is the flexibility of its respiratory chain. The human cell respiratory chain is composed of four respiratory enzymes with low mechanistic flexibility. On the other hand, the respiratory chain of P. aeruginosa contains 23 respiratory enzymes that ensure survival under harsh conditions. To elucidate the physiologic role of these respiratory enzymes, our research compared the growth parameters of wild type P. aeruginosa and nine separate respiratory enzyme mutants, in both LB and artificial urine media (AUM). The deletion mutants include the sodium-translocating NADH: quinone oxidoreductase, complex I, succinate dehydrogenase, cytochrome bc1 complex, cytochrome c oxidase and cyanide insensitive terminal oxidase. Our data indicate that the growth curve of the cytochrome bc1 complex knockout mutant showed a significantly lower yield and lower growth rate compared with the wild type in both LB and AUM media. Additionally, the cyanide insensitive terminal oxidase mutant showed a significant lower yield compared with the wild type in LB media growth. These results indicate the important roles of these enzymes in the cell biology of P. aeruginosa.
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Title: MITOCHONDRIA RELOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTED HFF-1 CELLS
Creator: Shuppara, Alexander Mitchell
Date: 2021
Description: Chlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases...
Show moreChlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases worldwide, C. trachomatis is the most prevalent sexually transmitted infection. It manifests as either trachoma, lymphogranuloma venereum, or other urogenital tract sequelae. As an intracellular pathogen, Chlamydia must scavenge for essential metabolites from establishing networks with its host’s organelles including Golgi apparatus, endoplasmic reticulum, endocytic vesicles, mitochondria, and the cytoskeleton. C. trachomatis was considered an “energy parasite” that is entirely dependent on their host’s ATP production. Yet, recent mitochondrial inhibitor-based evidence suggests that C. trachomatis possess a sodium-based energy gradient for ATP production. Despite this finding, literature on specific interactions between host cell mitochondria and C. trachomatis requires further definition. This project evaluates mitochondrial dynamics changes from C. trachomatis infection in the human foreskin fibroblast cell line, HFF-1. We first defined C. trachomatis growth characteristics in HFF-1 over 36 hours-post infection. Next, we determined changes in mitochondrial dynamics and content throughout infection using immunofluorescent and immunoblotting techniques. observations on infected cells show mitochondrial morphology changes from an elongated appearance at the early stages of infection to fragmented in the late infection stages. Unlike in HeLa cells, HFF-1 remains in a normal distribution throughout the cell and we do not observe mitochondria relocalizing toward the inclusion. By studying mitochondrial relocalization dynamics, new insights into the dynamic and parasitic relationship of Chlamydia and its host can be discovered.
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Title: SALMONELLA SURVIVAL AND TRANSCRIPTOMIC RESPONSE ON FRESH-CUT CANTALOUPE FLESH WITH AND WITHOUT ORGANIC ACID PRETREATMENT
Creator: Zhou, Xinyi
Date: 2020
Description: Outbreaks of Salmonella enterica associated with fresh-cut melons are becoming more frequent in recent years. Antimicrobial activity of...
Show moreOutbreaks of Salmonella enterica associated with fresh-cut melons are becoming more frequent in recent years. Antimicrobial activity of organic acids on fresh-cut melons have been previously studied. However, little is known about the molecular mechanism behind the antimicrobial activity of organic acid.Four strains of S. enterica were utilized: Newport 36796 and 339652, and Typhimurium LT2 and 46249. Both high and low inoculation levels were performed. For low level, each strain was individually cultured and spot-inoculated onto separate 100 g untreated fresh-cut cantaloupe samples resulting in 4 log-CFU/g. For high level, samples were first submerged into 2% citric acid or malic acid for 1 minute or left as untreated control. Cantaloupe were spot inoculated with one of four strains which resulted in 7-log CFU/g. All inoculated samples were air-dried for 1 h then stored at 4°C for 7 d in deli containers. Enumeration was conducted at 0, 1, 3, 5, and 7 d. Duplicate samples were used in each of three independent trials and results were analyzed by Student’s t-test, p≤0.05. Samples for sequencing were prepared using the TruSeq Stranded mRNA kit and run on a MiSeq according to the manufacturer instructions.For low inoculation level, population of all four strains increased significantly from 0 to 3 d. The two cantaloupe outbreak-related strains (Newport 339652 and Typhimurium 46249) increased significantly between 0 and 7 d from 3.44±0.11 to 3.76±0.13 and 3.36±0.12 to 3.78±0.19 log CFU/g, respectively. For high inoculation level, the population on the untreated cantaloupe was 6.55±0.18 log CFU/g at 7 d, whereas it was significantly lower on the citric and malic acid-treated cantaloupes (6.26 ± 0.09 log CFU/g and 6.07 ± 0.18 log CFU/g). After 1 d, S. enterica genes were downregulated up to 437.4-fold compared and upregulated up to 23.2-fold. The notable downregulated genes encoded proteins related to catalyzing metabolism (L-aspartate oxidase) and also related to nutrient uptake (PstC).The results of this study can aid in understanding population dynamics of S. enterica on fresh-cut cantaloupes and efficacy of malic and citric acids. The results can also aid in understanding mechanism underlying S. enterica survival on fresh-cut cantaloupes.
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Title: SODIUM-BASED ENERGY METABOLISM AND DYNAMIC ENERGY DEPENDENCY OF CHLAMYDIA TRACHOMATIS
Creator: Liang, Pingdong
Date: 2019
Description: Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for various human diseases including trachoma, genital tract...
Show moreChlamydia trachomatis is an obligate intracellular bacterium that is responsible for various human diseases including trachoma, genital tract infections, and lymphogranuloma venereum. Energy metabolism consists many essential pathways to generate energy for every organism. However, it remains much unknown in C. trachomatis. For decades, C. trachomatis has been considered as an “energy parasite”, which needs the energy supply from the host cells entirely. In contrast, genomic studies show that this bacterium is capable of encoding enzymes that involve energy metabolism. However, no experimental data were provided to support the genomic information due to the peculiar developmental cycle of C. trachomatis inside the host cells. In this project, the oxidative phosphorylation pathway of C. trachomatis is first identified with experimental results. This pathway starts with the sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) transferring the electrons along the respiratory chain and generating a sodium gradient across the membrane. C. trachomatis contains an A-type ATPase that can utilize this sodium gradient to generate ATP. In vitro experiments in mammalian cells with different respiratory inhibitors show that C. trachomatis is not an obligate energy parasite. Instead, it has a dynamic energy dependency on the host metabolism that the bacterium switches from entirely to partially relying on the host energy metabolism for its energy requirement. The sodium gradient established by NQR and/or other transporters is of great importance to chlamydial metabolism. Further, the respiratory inhibitors test on interferon-γ-induced persistence of C. trachomatis in mammalian cell cultures shows that an inhibited energy metabolism prevents and eliminates the persistent form. This study provides new insights about antibiotics development and therapeutic methods against C. trachomatis infections.
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Title: Establishing Bisphenol A Degradation and Enhancing Microbial Fuel Cell Performance by Biofilm Optimization of Shewanella Oneidensis MR1
Creator: Zhou, Jiacheng
Date: 2023
Description: Bisphenol A (BPA) has been widely used as a plasticizer in the production of synthetic polymers, such as those used in food storage containers...
Show moreBisphenol A (BPA) has been widely used as a plasticizer in the production of synthetic polymers, such as those used in food storage containers and bottles. However, BPA interferes with endocrine systems, causing carcinogenicity, immunotoxicity, and embryotoxicity. Biological water treatment processes scarcely remove BPA, owing to the poor BPA degradability and efficiency of the applied microorganisms. Shewanella oneidensis has been studied and used for the biodegradation process in wastewater treatment because of its excellent extracellular electron transfer properties. In this work, we engineered S. oneidensis MR1 to enable BPA degradation by producing ferredoxin (Fdbisd) and cytochrome P450 (P450bisd) originating from Sphingomonas bisphenolicum AO1. The engineered S. oneidensis exhibited a higher BPA degradation efficiency than that of Escherichia coli producing the same enzymes. The endogenous ferredoxin and ferredoxin reductase of S. oneidensis participated in BPA degradation, and overexpression of mtrC, omcA, and So0521, which encode S. oneidensis cytochromes, decreased BPA. We developed BPA-degrading S. oneidensis biofilms. We measured these optimized BPA-degrading S. oneidensis biofilm in a single chamber microbial fuel cell formed on different carbon electrodes by morphology. Cyclic voltammetry and electrochemical impedance spectroscopy were measured to analyze the biofilm-electrode performance. The biofilm colonization was also measured by confocal laser scanning microscope and scanning electron microscope. And the developed microbial fuel cell was used to degrade BPA and the biofilm developed on different type of carbon anodes was identified. This study provides insights into biocatalyst utilization for the biological degradation of toxic organic compounds.
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Title: Examination of Power Ultrasound and Organic Acid-based Hurdle Technology in the Reduction of Salmonella Enterica on Peaches and Apples
Creator: Mathias, Hina Valida
Date: 2023
Description: Fresh produce includes fruit matrices like whole peaches and apples that are minimally processed and are a popular choice among different...
Show moreFresh produce includes fruit matrices like whole peaches and apples that are minimally processed and are a popular choice among different types of demographics because of their nutrition content and health benefits. However, there have been increasing pathogen outbreaks in these matrices over the past few decades, which are majorly rooted in cross contamination either due to poor handling pre and post processing or the insufficient reduction of the pathogen at processing by the applied hurdle technology. While chemical sanitizers are a popular option in the food industry, the awareness and demand for green consumerism and sustainability have created a need for research to determine the efficacies of organic acids and non-thermal technologies like power ultrasound in the reduction of different pathogens on different food matrices. This study focusses on the S. enterica reduction capabilities of three organic acids – citric, malic, and lactic alone and in combination with 40 kHz power ultrasound at 1, 2 and 5% for treatment times of 2, 5 and 10 min on whole yellow peaches and gala apples. Peaches and apples were spot inoculated with a four-strain cocktail of S. enterica, resulting in 9 log CFU/fruit. Post air drying for 1 h, the fruits were treated with water, 1, 2, or 5% citric, lactic, or malic acid for 2, 5 or 10 min with and without power ultrasound treatment at 40 kHz. The population of S. enterica on the fruits was enumerated before and after treatment. Three independent trials with triplicate samples were performed for each condition. Population differences were evaluated via Student's t-test and ANOVA; p<0.05 was considered significant. The initial level of inoculum ranged from 8.67 ± 0.41 to 8.20 ± 0.26 log CFU/peach and 7.28 ± 0.60 to 8.17 ± 0.37 log CFU/apple in peaches and apples, respectively. Water treatments showed pathogen reduction as high as 1.22 log CFU/peach and 1.02 log CFU/apple. Citric acid treatments on peaches showed significant pathogen reduction at higher time increments at 5% with a reduction of S. enterica as high as 2.24 log CFU/peach after 10 min. Malic acid showed the highest recorded log reduction in peaches at 5% and 10 min being 4.20 log CFU/peach (n=1/9, samples above the enumeration limit) and apples at 5% and 5 min being 3.71 log CFU/apple (n=4/9, samples above the enumeration limit) both in combination with an ultrasound. Lactic acid, unlike the other two organic acids, showed a pathogen reduction of over 3 log CFU/fruit at 2% after 10 min, with the highest pathogen reductions of 3.76 log CFU/peach and >3.62 log CFU/apple at 5% and10 min. There was no particular trend with significant enhancement of pathogen reduction either with time increment or the addition of ultrasound and varied with the varying acids, treatment conditions and fruit matrices.
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Title: Population dynamics and pathogens of the western bean cutworm (Striacosta albicosta)
Creator: Bunn, Dakota C.
Date: 2022
Description: Understanding an herbivorous pest’s population dynamics is necessary to ensure proper integrated pest management strategies are being...
Show moreUnderstanding an herbivorous pest’s population dynamics is necessary to ensure proper integrated pest management strategies are being developed and used. The western bean cutworm is a pest of both corn and dry beans that is understudied and difficult to manage due to its nocturnal lifestyle, adaptation to current management techniques and a general lack of understanding regarding its population structure. Our studies focused on the effects of host plant and pathogens on western bean cutworm population structure and found that mainly adults which developed on corn are contributing to the next generation of western bean cutworm in Michigan, making corn and dry beans unsuitable as co-refuges in insecticide resistance management strategies.We also found a 100% prevalence of the Nosema sp. in the adult population of western bean cutworm in Michigan. This prevalence, when paired with the consistent crop damage caused annually by the western bean cutworm, which confirms an abundance of cutworms are present, suggests the infection is slow acting and non-lethal to its host. Following sequencing, assembly, and annotation of the Nosema sp. genome, we were unable to provide a reason for the Nosema sp.’s low virulence, however, we were able to confirm the presence of all 6 polar tube proteins. Upon further examination of the Nosema sp. genome we were able to determine that it is a true Nosema with genome size of ~9.57 Mbp (~20% of which are transposable elements), which is within the typical range for this genus.
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Title: Factors Influencing the Level of Detection of Testing Listeria monocytogenes in Ice Cream
Creator: Chen, Bairu
Date: 2022
Description: The increasing evidence has shown that having a sensitive detection method for Listeria monocytogenes in food products is critical for public...
Show moreThe increasing evidence has shown that having a sensitive detection method for Listeria monocytogenes in food products is critical for public health as well as industrial economics. L. monocytogenes was associated with foodborne illness outbreaks linked to ice cream in the United States from 2010 to 2015, with another recent outbreak under investigation. The FDA Bacteriological Analytical Manual (BAM) method was commonly used for L. monocytogenes detection. However, the performance characteristics of the chromogenic methods (MOX, RLM, and R&F agars) remain to be elucidated. The factorial effect on Level of Detection (LOD) as an essential element of the International Organization for Standardization (ISO) approach for qualitative method validation was investigated in this study.For examining the LOD of L. monocytogenes in ice cream, fractional contaminated samples were prepared with the ice cream obtained from the 2015 outbreak and enumerated using the FDA BAM Most Probable Number (MPN) method for Listeria. The effect of test portion size was determined by comparing 10g and 25g using the BAM method with chromogenic agars (MOX, RLM, and R&F). The ISO single-lab validation requirement was followed for the factorial effect study, including four different factors: sample size (10g and 25g), ice cream types (commercially available regular vanilla ice cream and vanilla ice cream with low fat and no added sugar), re-freezing process (with re-freezing and without re-freezing process), and thawing process (slow thaw and fast thaw). LOD and relative LOD (RLOD) were computed using MiBiVal software to compare the sensitivity of the three chromogenic agars and the different factors. For all of the detection experiments, presumptive colonies were identified using the API listeria kit. The 2015 naturally contaminated ice cream was enumerated and resulted in an average contamination level of 2.15 MPN/g. At fractional levels of 0.25 MPN/10g and 0.75 MPN/10g, the positive rates of L. monocytogenes detected from 10g and 25g of sample portions were consistent with the statistically theoretical positive rates. The RLOD values for the reference method (MOX) and the alternative methods (RLM, R&F) were above 1 in both portion sizes, which suggested that MOX was slightly more sensitive than RLM and R&F. The factorial effect study indicated that the four factors have no significant influence on the LOD of L. monocytogenes detection at the fractional contamination levels. However, the test portion size of 25g provided more consistent results among the chromogenic media than the 10g portion size. Fat content was shown to have an effect on L. monocytogenes detection in a large test portion. The information from this study will be useful for the improvement of the reproducibility of a qualitative detection method and can also be used for data analysis standards such as ISO 16140 in method validation studies.
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Title: Efficacy of Organic Acid Treatments for the Reduction of Listeria Monocytogenes on Hard Boiled Eggs
Creator: Khouja, Bashayer
Date: 2022
Description: Ready-to-eat hard-boiled eggs (HBEs) are a popular and convenient choice for consumers and food servicers. Recentrecalls of hard-boiled eggs...
Show moreReady-to-eat hard-boiled eggs (HBEs) are a popular and convenient choice for consumers and food servicers. Recentrecalls of hard-boiled eggs have highlighted the susceptibility of contamination with Listeria monocytogenes. HBEs are generally treated with antibacterials to ensure the safety and quality of the product. While citric acid is often used, research has determined it is not effective in some situations; therefore, the assessment of additional organic acids is necessary. This study examined the efficacy of acetic, lactic, and malic acid on the reduction of L. monocytogenes on HBEs after a 24- hour treatment trials and 28 days storage trials. Fresh eggs were cooked in boiling water, peeled, and stored at 4°C for 24h before use. For treatment trials, HBEs were dip- inoculated with a 4-strain cocktail of rifampicin resistant L. monocytogenes, resulting in 8 log CFU/egg. Following air drying, hard-boiled eggs were treated at 5 or 25°C with 2% acetic, lactic, or malic acid. L monocytogenes populations were enumerated in intervals up to 24h by homogenization of HBEs with BLEB and cultivation on BHIrif. For pre- treatment storage trials, HBEs were first dip- inoculated with a rifampicin- resistant 4- strain L. monocytogenes cocktail for 20 min, resulting in 1 log CFU/egg, air dried for 10 min, followed by treatment with 2% acetic, lactic, or malic acid for 24 h at either 5 or 25°C. For post- treatment inoculation trials, HBEs were first soaked in 2% acetic, lactic, or malic acid for 24 h at either 5 or 25°C, air dried for 10 min, spot-inoculated at 1 log CFU/egg, and then dried for 20 min. All HBEs were individually stored in bags at 5°C for up to 28 days. The presence of L. monocytogenes was determined at intervals during storage by enrichment with BLEB on BHIrif and Brilliance Listeria Agar. Triplicate eggs were assessed for each timepoint, and three independent trials were conducted. Data were analyzed by Student’s T-test, ANOVA, and Fisher’s exact test, p≤0.05. The initial inoculation level of L. monocytogenes on HBEs was 8.27±0.37 log CFU/egg. After 24 h treatment, all L. monocytogenes populations were significantly reduced on HBEs. At 5°C, populations were reduced by 3.15±0.70, 3.46±0.02, and 4.78±0.23 log CFU/egg. Compared to 5°C, a significantly higher population reduction occurred with acetic and lactic acid when treatment occurred at 25°C. The inactivation of L. monocytogenes on HBEs for the storage trials was associated with the order of the contamination: pre-or post-the acid treatment. Prior storage, L. monocytogenes was detected on 100% of the HBEs. Malic acid pre-treatment was significantly effective in eliminating L. monocytogenes on HBEs at 5 and 25°C, while acetic acid was effective only at 5°C. All acids did not eliminate L. monocytogenes in the case of post-treatment contamination at any tested temperature. The results of this study aid in understanding the efficacy of organic acid treatments against L. monocytogenes on HBEs. Results are useful in the development of preventive controls and guidelines to ensure the safety of HBEs.
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Title: UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Creator: Fang, Kuili
Date: 2020
Description: Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm...
Show moreMany chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in mono-species biofilms. The first project (Chapter 3) investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC mono-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a pathogen causing the outbreaks of hemorrhagic colitis. Conventional antibiotics treatment is not recommended for EHEC infection as antibiotics trigger Shiga toxin production of EHEC and aggravate hemolytic-uremic syndrome. EHEC biofilm formation is closely associated with its virulence expression. Previously, we identified that probiotic E. coli Nissle 1917 (EcN) secretes DegP resulting in the inhibition of EHEC biofilm formation in a dual culture. DegP is a serine protease exhibiting both proteolytic and chaperone functions and binds to outer membrane proteins (OMPs) of target cells. However, the extracellular function of DegP is not clear. We hypothesized that binding of DegP to OMPs of EHEC might inhibit EHEC biofilm formation by affecting the adhesion ability or changing biofilm-related gene regulations of EHEC. We constructed EHEC mutants lacking ompA, ompC, or ompF individually and in combination and assessed their biofilm formation in the presence of DegP-secreting EcN in the co-culture or by adding purified DegP. It was found that both ompA and ompC double deletion decreased EHEC single species biofilm, and also caused that DegP inhibited more EHEC biofilm (about 25 fold inhibition) than DegP inhibited EHEC wt biofilm (about 10 fold), indicating that OmpA and OmpC are more related to EHEC biofilm than OmpF, and OmpA and OmpC might deplete DegP inhibitory functions. On the other hand, DegP S210A, a DegP mutant lacking protease function, inhibited EHEC wt biofilm, indicating that DegP’s biofilm inhibition function is not from its protease activity. Additionally, EHEC transcription profiles in the presence of DegP showed that DegP up-regulated expressions of cellulose production related genes (csgD and bcsA) and motility related genes (flhD, qseB), which were all involved in EHEC biofilm inhibition, and down-regulated Shiga toxin 2 virulence gene (stx2). Besdies, DegP promoted EHEC cellulose production and motility, which is consistent with transcription profile, and Shiga toxin 2 production will be further tested. This study reveals a new function of DegP secreted by EcN in controlling biofilms and leads us to develop an alternative strategy to control biofilm-related infections. Foodborne pathogen Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the Chapter 5 study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 times 10^6 CFU/cm2 L. monocytogenes ATCC19115 in single-species biofilms to 1.1 times 10^5 CFU/cm2 in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (> 30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.
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Title: Development of validation guidelines for high pressure processing to inactivate pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in treated juices
Creator: Rolfe, Catherine
Date: 2020
Description: The fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional...
Show moreThe fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional juice division is cold pressure, which refers to the application of high pressure processing (HPP) at low temperatures for a mild treatment to inactivate foodborne pathogens instead of thermal pasteurization. HPP juice manufacturers are required to demonstrate a 5-log reduction of the pertinent microorganism to comply with FDA Juice HACCP. The effectiveness of HPP on pathogen inactivation is determinant on processing parameters, juice composition, packaging application, as well as the bacterial strains included for validation studies. Unlike thermal pasteurization, there is currently no consensus on validation study approaches for bacterial strain selection or preparation and no agreement on which HPP process parameters contribute to overall process efficacy.The purpose of this study was to develop validation guidelines for HPP inactivation and post-HPP recovery of pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in juice systems. Ten strains of each microorganism were prepared in three growth conditions (neutral, cold-adapted, or acid-adapted) and assessed for barotolerance or sensitivity. Pressure resistant and sensitive strains from each were used to evaluate HPP inactivation with increasing pressure levels (200 – 600 MPa) in two juice matrices (apple and orange). A 75-day shelf-life analysis was conducted on HPP-treated juices inoculated with acid-adapted resistant strains for each pathogen and examined for inactivation and recovery. Individual strains of E. coli O157:H7, Salmonella spp., and L. monocytogenes demonstrated significant (p <0.05) differences in reduction levels in response to pressure treatment in high acid environments. E. coli O157:H7 was the most barotolerant of the three microorganism in multiple matrices. Bacterial screening resulted in identification of pressure resistant strains E. coli O157:H7 TW14359, Salmonella Cubana, and L. monocytogenes MAD328, and pressure sensitive strains E. coli O15:H7 SEA13B88, S. Anatum, and L. monocytogenes CDC. HPP inactivation in juice matrices (apple and orange) confirmed acid adaptation as the most advantageous of the growth conditions. Shelf-life analyses reached the required 5-log reduction in HPP-treated juices immediately following pressure treatment, after 24 h in cold storage, and after 4 days of cold storage for L. monocytogenes MAD328, S. Cubana, and E. coli O157:H7 TW14359, respectively. Recovery of L. monocytogenes in orange juice was observed with prolonged cold storage time. These results suggest the preferred inoculum preparation for HPP validation studies is the use of acid-adapted, pressure resistant strains. At 586 – 600 MPa, critical inactivation (5-log reduction) was achieved during post-HPP cold storage, suggesting sufficient HPP lethality is reached at elevated pressure levels with a subsequent cold holding duration.
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