Application of oregano oil nano-emulsions to inhibit foodborne pathogens on bagged lettuce

Kanika Bhargava, Denise S. Conti, Sandro R.P da Rocha and Yifan Zhang, Wayne State University, Detroit, MI 48202

Although the antimicrobial activities of essential oils such as oregano are well documented, there are limited efforts in addressing the challenges associated with their application in fresh cut produce. Oregano oil emulsions, the potential natural sanitizers for the produce industry and consumers were formulated and evaluated for their efficacy in inhibiting the growth of common foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes) on lettuce. Emulsions with two concentrations of oregano oil (0.05 and 0.1%) were prepared by sonication utilizing food grade emulsifier, Tween 80 and characterized by dynamic light scattering technique and atomic force microscopy. Lettuce was artificially inoculated with the above mentioned strains followed by treatment in emulsions for one minute. Samples were stored at 4C and enumerated at fixed interval (0, 1 and 3 days) on selective media. The average diameter of nano-emulsions was 197.9nm and the oil droplets were round, uniformly distributed and not aggregated. Compared to the water control, lettuce showed between 3.0-3.6-log, 3.1-3.2-log and 2.0-2.4-log CFU/g reductions in L. monocytogenes, S. Typhimurium and E. coli O157:H7 by day 3, respectively. The initial bacterial load was reduced by oregano oil emulsions and maintained during treatment. The application of antimicrobial nano-emulsion of oregano oil is a simple and effective preservation method for ready to eat bagged lettuce.

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Differentiating Live from Dead: Verification of Ballast Water Treatment Technologies Using Fluorescein Diacetate

Eboni A. Reed, Nataliya Sachovska, Jason P. Gizicki and Jeffrey L. Ram, Wayne State University, Detroit, MI

Background: To reduce the number of non-native species trans-ported into the Great Lakes incipient international and U.S regulations will require ship ballast water treatment (BWT) to decrease viable organisms before its discharge. BWT is predicted to eliminate the source of at least half of the non-native species that might enter the Great Lakes. This project tested fluorescein diacetate (FDA), a membrane permeable fluorogen substrate of hydrolytic enzymes of bacteria and plankton, as a way to differentiate live from dead organisms in ballast water.
Methods: Plankton cultures and other samples (river, ballast water) were filtered using a syringe filtration apparatus, backwashed off the filter into a small volume of Jaworski’s medium, pH 7, and assayed in a fluorescence plate reader. Assays were performed on cultures with and without chlorine (3 mg/L, 3 hours) or heat (30 minutes, 95C) treatment. In addition, whole samples of river and ballast water were separated by size, and assayed.   
Results: Experiments revealed linearity between relative amount of fluorescence and time over 60 minutes, as well as relative amount of fluorescence and the amount of culture within a sample (r2=0.92, n=2). After plankton and ballast water had been treated with heat or chlorine, respectively, the fluorescence production was low in comparison to untreated samples (1.8% heat treated, 5.9% chlorine treated). For ballast water samples accompanying microbiological tests showed that chlorine effectively reduced the number of live coliform & E. coli to <1 per 100 mL. Samples separated by size yielded less fluorescent product (about 20% of the total) for organisms greater than 10 ?m, in comparison to smaller organisms (n=3).
Conclusions: The FDA method is useful in distinguishing live versus dead organisms, and can be used to assess organisms in ballast water, and the effectiveness of ballast water treatment. This method is capable of detecting organisms over a broad size range. The future plan is to compare two methods of verifying ballast water treatment, FDA and PMA-PCR (a DNA-based live/dead method), when organisms are separated by size.

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Further Studies of Inositol Derivatives

Mary J. Thwaites, Petra  R. Kohler, and Silvia Rossbach, Department of Biological Sciences, Western Michigan University, Kalamazoo MI, 49008

Nitrogen is a limiting nutrient in agricultural settings; crops generally require nitrogen supplementation in the form of fertilizer. While molecular nitrogen is the most abundant element in the earth's atmosphere, it occurs in a chemically inert form due to the strong triple bond between the nitrogen atoms. To remedy this problem humans generate artificial fertilizers using the Haber-Bosch process. Unfortunately this is very costly and consumes 1-2% of the world's total energy supply each year. An alternative to this expensive process is biological nitrogen fixation of atmospheric nitrogen into ammonia. This process is carried out by free-living or plant-associated bacteria. Sinorhizobium meliloti is one of the plant-associated nitrogen fixing bacteria; these organisms actually infect the plant and form specialized organs known as root nodules. In this nodule the bacteria use the enzyme nitrogenase to fix atmospheric nitrogen for use by the host plants, and in return the plants supply the bacteria with carbon sources. One strain of S. meliloti, L5-30, has been discovered to synthesize compounds known as rhizopines. Rhizopines are inositol derivatives including scyllo-inosamine and L-3-O-methyl- scyllo-inosamine. Rhizopines play a role in competition for nodulation and have been proposed for the selection of beneficial microorganisms that can catabolize these compounds. Only about 10% of S. meliloti strains can use rhizopines as their sole source of carbon; these strains have a cluster of genes known as the moc genes. The mocABC and mocR genes are required for catabolism of scyllo-inosamine and two more genes, mocD and mocE, are required for catabolism of L-3-O-methyl- scyllo-inosamine. Genes homologous to mocD and mocE are also found in the genome of S. meliloti strain 2011, which was used in this study.  The purpose of this research was to elucidate the function of these genes using rhizopine-like compounds.

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Identification and Analysis of Breaks in Human Common Fragile Sites Using Saccharomyces cerevisiae

Will Fitzsimmons and Anne Casper, Eastern Michigan University, Ypsilanti, MI 48197

It is important to understand the genetic changes causing uncontrolled cell proliferation in tumors.   Common fragile sites, areas of high instability in the genome, are frequently altered in tumor cells.  These sites can break under conditions of replication stress, resulting in chromosomal aberrations and loss of gene function. Two hypotheses have been proposed to explain why common fragile sites break.  First, A-T rich segments with high flexibility sequences in the fragile site may fold into secondary structures.  These structures arise when replication stress results in long regions of single stranded DNA at the replication fork, allowing intra-strand base pairing.  These secondary structures cause replication fork stalling and fork collapse, resulting in chromosomal breakage at the site of the secondary structure.  Another hypothesis proposes that fragile site regions lack sufficient origins of replication, which paired with replication stress results in failure to complete replication.  Breaks will result in locations farthest from the origins, due to un-copied chromosomes separating during mitosis. We are testing these two hypotheses by mapping break locations in two yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae with human DNA inserts.  One YAC contains an 852 Kb segment containing fragile site FRA3B, and a separate YAC contains a 161 Kb segment of FRA7H. Among 30 broken YACs with the FRA3B insert, we have found that under conditions of replication stress, breaks mainly occur within a small region towards the end of the insert. Under the same conditions, 30 FRA7H breaks are located toward the middle of the FRA7H fragile site region.  Having different areas of concentrated breaks for each fragile site supports the hypothesis that sequence structure plays the main role in fragile site breakage, due to the fact that breaks occur at different distances from the origin of replication. Mapping of the exact break locations, to determine whether they correspond with locations containing flexible sequences, would lend further support to this hypothesis.

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Degradation of Tetracycline by Rhizospheric Bacteria Recovered from an in vitro Hydroponic Phytoremediation System

Aparupa Sengupta, Andrew Kennedy, Mike Potes, Rupali Datta, and Susan Bagley, Michigan Technological University, Houghton, MI 49931

Antibiotics are emerging contaminants worldwide.  Due to insufficient policy regulations and public awareness, constant exposure of antibiotics in environmental sources has created a major environmental concern.  The Waste Water Treatment Plants are also not equipped to filter-out these compounds before the discharge of the disinfected effluent into water sources (lakes and streams).  The aim of this study is to identify possible roles of rhizhosperic bacteria in the Tetracycline (TC) degradation process and to optimize the process. In our lab, a hydroponic remediation system has been developed using vetiver grass (Chrysopogon zizanoides L. Nash) to remediate tetracycline (TC) from water.  The remediation results suggest the successful uptake of TC by vetiver grass.  Also, the degradation of TC in the hydroponic tanks with no plants suggests that there is a role of rhizospheric bacteria in the degradation process.  Hence, TC-tolerant bacteria were recovered from vetiver roots and contaminated water during the 30-day period of remediation using R2A agar with different levels of TC (10, 25, 50 and 100 ppm).  Ten TC-tolerant bacterial isolates were selected for further study.  The sampling period was based on the growth study results. Day 0 was selected to establish the baseline, day 1 was the pre-log phase, day 2 was the log phase, and day 21 shows the complete degradation of TC.  Two different kinds of media were used to show difference in types of metabolism i.e., in a general purpose medium (TSB) with TC and a minimal medium (MSM) with TC as the single carbon source.  Results were obtained using a Spectrophotometer, HPLC and MS-MS. Results indicate that some of these TC-tolerant bacteria can use TC as their sole carbon source.  These bacteria transform TC into its isomers anhydro-TC, epi-TC and other unknown metabolites establishing their role in TC degradation.  The bacterial strains that showed highest degradation rate of tetracycline (in medium with 50 ppm of TC) were selected for optimization study (pH, temperature and oxygen) to increase the rate of degradation.

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Automated Assessment Of Microscopic Organisms In Environmental Samples Using Fluorescein Diacetate

Ahsan Akram, Sukhdeep Singh, Jeffrey Ram
Dept. of Physiology, Wayne State University, Detroit, Michigan

Background: The experiments tested whether fluorescein diacetate (FDA) (see Reed et al.) can be used in an automated fluorescence detection system to assay live organisms in environmental or ballast water samples. Hydrolytic cleavage of FDA (colorless) produces fluorescein (fluorescent). Methods: The prototype automated system pumped water samples through a filter and subsequently backwashed with buffer and FDA into a 3 mL cuvette. Fluorescein emission (515-530 nm) was measured by a fiber optic spectrometer for 10-30 minutes. The reuse of filters and efficiency of cleaning by alternately backwashing with deionized (DI) water was tested manually and assayed on a fluorescent plate reader.  Experiments tested B-PER as an anti-biofilm reagent. Biofilm was created in cuvettes through overnight incubation of phytoplankton or water. A computer-controlled relay board operated valves and pumps and fluorescence data were recorded with real-time remote control and monitoring of results using TeamViewer software. Results: FDA-hydrolysis in environmental water samples increased linearly over time. Fluorescence in DI water assays was 7.0±8.3% of the average intensity of environmental water samples (n=7). Cellulose acetate 0.2 μm filters could be reused 20 times; nylon filters failed after 6 cycles. Backwashing with >60 mL of DI water cleaned the filter efficiently for reuse. Filters reused with DI, 60%, and 90% strength Detroit River water samples yielded average intensities of 64±5, 694±45, and 1161±21, respectively over many repeats in a typical experiment. B-PER (2%, 10 min; 10%, 10 min; 10%, 20 min) removed phytoplankton biofilm residual FDA hydrolysis activity by 25% (n=1), 75% (n=2), and >90% (n=1), respectively. Environmental samples measured after B-PER treatment (10%, 20 min) followed by DI rinse was 107.2% and 98.4% of corresponding pre-B-PER measurements (n=2). Conclusions: These studies demonstrated that automated measurements of FDA hydrolysis with a reusable filter backwash system under computer control should be applicable to remote-controlled monitoring of live organisms in ballast water (provisional patent pending).

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The Correlation Between Origin Paucity and Fragile Site Breaks Exposed to DNA Replication Stress

Katherine A. Dziuba and Anne M. Casper, Eastern Michigan University, Ypsilanti, MI

Under conditions of replication stress, areas of high genomic instability, known as fragile sites, tend to fragment or break. These breaks can result in deletions within important tumor suppressor genes at fragile site regions. It has been proposed that a regional lack of replication origins on the chromosome in fragile site regions is responsible for breaks. With fewer origins present, each DNA replication fork has to travel a very long distance for DNA replication to be completed. Any delay of DNA synthesis would lead to incomplete DNA replication in the fragile site region before the cell enters mitosis. If the cell does not completely replicate DNA, then the chromosome may break at the fragile site when sister chromatids are separated in the process of mitosis.
        Our lab has conducted previous research on fragile site FRA3B, located within the FHIT tumor suppressor gene on human chromosome 3. Using a yeast artificial chromosome (YAC) in the yeast, Sacchraromyces cerevisiae, which contains 1.3 Mb of human sequence with a portion of the human fragile site FRA3B, and several gene markers, we determined that breaks within the fragile site are common in intron 5 of the FHIT gene. The YAC has only one origin of replication. To test the lack of origins hypothesis, we are working to insert another origin on the YAC, near the site of the breaks. To insert the second origin, we have modified the YAC to insert pCORE, which is a selectable marker. Next we will replace pCORE with the origin site. After insertion of the second origin site, we expect to see breaks occurring near the center or towards the front of the DNA insert. These regions are furthest away from the origin sites and are believed to experience delayed DNA replication that leads to breaks. Due to the presence of two origin sites, it is also possible that DNA replication will occur efficiently and no breaks will be observed.

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Dose Response analysis of Pseudomonas aeruginosa: an opportunistic pathogen

Sushil B. Tamrakar and Joan B. Rose, Department of Fisheries and Wildlife, Michigan State University, Michigan

Pseudomonas aeruginosa is a Gram-negative, aerobic rod shaped free living bacterium belonging to the bacterial family Pseudomonadaceae. It is an opportunistic pathogen which may cause infection to immunocompromised and immunosuppressed person. Human diseases caused by P. aeruginosa are: i) bacteremia in severe burn cases, ii) chronic respiratory tract infection in cystic fibrosis patients and iii) ulcerative keratitis in users of soft contact lenses.  The main objective of this study is to analyze the dose-response relationship of P. aeruginosa in a host and its response to pathogen. We also tried to analyze the data from open literature that could describe the common infections in human.  Suitable data sets were explored from open journal articles. Hazlett et al. (1978) studied the susceptibility of newborn and infant mice to eye infection by P. aeruginosa. In one experiment, the pathogen was inoculated under unopened eyelids of infant mice in absence of prior corneal wounding. In the second experiment 15 to 16 day old mice were inoculated with bacteria on to wounded or unwounded corneas. The death due to bacteremia and infection (ketaritis) were recorded. Lawin-Br?ssel et al. (1993) studied soft contact lens (SCL) related eye infection in rabbits.

The dose-response model indicated the young mice (5 day old) were exponentially susceptible to death due to bacteremia. However, 10 day old mice showed slightly shallow response that 5 day old mice. However, none of the 15-16 day old mice died due to bacteremia and got only corneal keratitis in some mice. The data of 15-16 day mice were fit to exponential dose-response model. As suggested by Lawin-Brussel et al.(1993), the likelihood of developing keratitis depends on the amount of bacterial concentration. In an average, a dose of 1 CFU may cause death of 1/10000 exposed population due to bacterimia. An avarage dose of 0.26 CFU may cause ulcerative keratitis of 1/10000 among extended contact lens users.

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Determining DNA Break Points in the Human Fragile Site on Chromosome X

Catherine Kaminski and Anne Casper, Eastern Michigan University, Ypsilanti MI, 48197

Within all human chromosomes, there are regions of instability referred to as common fragile sites. These are areas within DNA that are more susceptible to breaks when cells are under replication stress. Fragile sites vary in length, but most are large, between 200-500 kilobases long. This raises concern because some fragile site regions contain important tumor suppressor genes, that can potentially be lost when there is a deletion or translocation of the chromosome at the fragile site, resulting in the proliferation of tumors. There are two hypotheses for why fragile sites may break when under replication stress. The first reason could be a lack of DNA replication origins within the fragile site region. This can result in unfinished replication by the end of mitosis, and this unfinished DNA would consequently be broken when sister chromatids separate in anaphase. The second hypothesis is that particular sequences of nucleotides in fragile site regions could form secondary structures such as hair-pin loops in the single-stranded DNA that exists at the replication fork. These structures can disrupt the course of DNA replication and can be cleaved off and lost during DNA repair. We are conducting experiments using the fragile site FRAXB on human chromosome X. Using a Yeast Artificial Chromosome (YAC), the fragile site is inserted into our YAC and replication stress is induced. With our experimental procedure we will be determining the sequence and location of breaks in relation to origins of DNA replication in approximately 30 independent clones. From our results we will be able to further evaluate these two hypotheses.

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Virulence signaling of the Streptococcus pyogenes salivaricin locus

Hannah M. Rowe, Phanramphoei Namprachan-Frantz and Melody N. Neely, Wayne State University School of Medicine, Detroit MI

This work was also presented at the 2012 Midwest Microbial Pathogenesis meeting and the 2012 International Gram Positive Pathogens meeting.

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Verifying Ballast Water Treatment

Natalia Sachovska, Jason P. Gizicki, Jeffrey Henquinet#, Phyllis Green#, Jeffrey L. Ram, Dept. of Physiology, Wayne State University, Detroit, MI and #Isle Royale NP, Houghton, MI

Introduction of microorganisms by ballast water transport may be detrimental to Great Lakes ecosystems and human health. This project aims to develop a system for shipboard members to test the effectiveness of chlorine ballast water treatments (BWT). Standard microbiological tests (Quanti-Tray 2000) and PMA-PCR molecular analysis were employed. Methods: This project analyzed environmental, untreated, and Ranger III ballast collected before and after chlorine treatment going to and returnin g from Isle Royale in Lake Superior. The samples are filtered using a syringe-filter apparatus, treated or not treated with propidium monoazide (PMA), preserved with DNAzol, and backwashed off the filters for analysis. PMA crosslinks DNA, preventing PCR amplification, only in organisms whose membranes have been damaged and are no longer considered living cells. The samples are analyzed quantitatively to determine the relative amounts of DNA amplifiable by universal bacterial and algal primers. Results: The PMA-PCR results indicate that the chlorine treatment is effective in significantly reducing the amount of bacteria and phytoplankton in Ranger III ballast water.  PMA-PCR data indicated a >150 fold decrease in live bacteria and about a >12.5 fold decrease in live phytoplankton between intake and discharge on two voyages of the Ranger III.  Microbiological tests indicate that chlorine reduced the number of coliforms and E. coli to <1 cfu per 100mL. PMA-PCR and microbiological analyses for the return voyage of the Ranger III have similar results.  Conclusion: The syringe filter method and PMA treatment is an effective means of discerning live from dead microorganisms and be can employed by shipboard members to test the effectiveness of their BWT system. This method is able to differentiate from dead and live microbes and bind specifically to the DNA of dead cell. Future plans include comparing the effectiveness of ultra violet light BWT treatment using the PMA-PCR method.

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