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SYNERGISTIC EFFECT OF FATTY ACIDS AND NISIN IN INHIBITING PERSISTER AND BIOFILM OF LISTERIA MONOCYTOGENES
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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Role of Respiratory Enzymes on Growth of Pseudomonas aeruginosa in Luria-Bertani and Artificial Urine Media
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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MITOCHONDRIA RELOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTED HFF-1 CELLS
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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SALMONELLA SURVIVAL AND TRANSCRIPTOMIC RESPONSE ON FRESH-CUT CANTALOUPE FLESH WITH AND WITHOUT ORGANIC ACID PRETREATMENT
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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Examination of Power Ultrasound and Organic Acid-based Hurdle Technology in the Reduction of Salmonella Enterica on Peaches and Apples
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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Factors Influencing the Level of Detection of Testing Listeria monocytogenes in Ice Cream
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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Efficacy of Organic Acid Treatments for the Reduction of Listeria Monocytogenes on Hard Boiled Eggs
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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