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Project Title: Genes involved in stress response of Listeria monocytogenes. Construction of mutant strains

University of Michigan investigator(s) and unit:  Cristina Bares, PhD, Assistant Professor of Social Work

International colleague(s) name and unit: Angélica Reyes-Jara PhD., Assistant Professor, Laboratory of Microbiology and Probiotics. INTA, University of Chile

Purpose: To evaluate the relevance of selected genes during the proliferation of L. monocytogenes under low temperature and copper exposure.

 Rationale: Foodborne diseases represent a major public health problem that globally produces large economic losses. The microorganisms causing these diseases can persist in animals reservoirs, survive in typical food industry environments and resist the conditions and technological processes, shaped precisely to control their growth. One of these pathogenic microorganisms is Listeria monocytogenes, which in recent years has been the causal agent of several outbreaks L. monocytogenes is a Gram positive, non-sporulating and ubiquitous microorganism that has the ability to grow at refrigeration temperatures. In humans, the consumption of food contaminated with L. monocytogenes produce listeriosis, characterized by a range of manifestations that ranges from febrile gastroenteritis to a more severe invasive disease [8, 9]. Although the incidence of listeriosis is low (0,29 cases per 100.000 population) it is considered a significant condition because its invasive form has a mortality rate of 30%. Listeriosis represents the third leading cause of death after infection with Salmonella and Toxoplasma. Invasive listeriosis predominantly affects newborns, pregnant women, the elderly and immune deficient patients.

In contrast to other foodborne pathogens, the strategy in food industry of applying low temperature to avoid bacteria proliferation does not work for L. monocytogenes, because it is able to overcome low temperature stress after an acclimation stage (transient arrest of cell growth). Once adapted to the new condition the bacterium proliferates again. This response is known as psychrotolerance.

To prevent food contamination with L. monocytogenes, the food industry applies “combinations of stressors” (known as hurdle technologies) and is always searching for alternatives that allow the control of the pathogens. In this regards antimicrobial properties of copper may constitute an effective alternative. Antimicrobial properties of copper were accepted for the first time in 2008 by the United States Environmental Protection Agency. Copper surfaces can eliminate bacteria like S. aureus, E. aerogenes, MRSA and Pseudomona aeruginosa that usually cause nosocomial infections. Recent studies also proved the efficacy of copper to control other microorganisms such as viruses, fungi and foodborne pathogens. Thus, copper appears to be an excellent alternative to control L. monocytogenes in the food industry.

We had evaluated the global transcriptional response of L. monocytogenes to both copper and low temperature. These results showed an early response an acute stress by copper. We presented evidence of a strong down-regulation of genes associated to membrane biosynthesis, metabolism of carbohydrates and flagella biosynthesis, which is most likely due to energy preservation efforts that allow bacterium survival. This research gives insight into use of copper to control contamination by L. monocytogenes. Finally, to evaluate the implication of the gene regulation over the synergistic effect we will delete specific genes (mutant design) and analyze its effect over copper homeostasis and virulence of L. monocytogenes strain.

Study design and methods:

  • To work in protocol for construction of Listeria monocytogenes mutants
  • To characterize bacterial mutants regarding to copper homeostasis
  • To characterize bacterial mutants regarding to virulence by gene expression quantification

Anticipated undergraduate/graduate st1udent activities on project:

  1. To organize the results already obtained from the water maze test.
  2. To analyze the data and obtain the results.
  3. To run statistical analysis and compare the groups.

Techniques/methods students should become familiar with in advance:

The student will be participating in cloning activities, DNA extraction protocol and PCR analysis.

Techniques/methods students should become familiar with in advance:

  • Handling of bacterial cultures,
  • In silico analysis,
  • Bacterial transformation,
  • PCR,
  • Cloning

Suggested readings (minimum of 3-5 articles): all articles are available for interested students. 

  • Cordero N, et al. 2016. Different Transcriptional Responses from Slow and Fast Growth Rate Strains of Listeria monocytogenes Adapted to Low Temperature. Front Microbiol 7:229.
  • Latorre M, et al. 2015. Synergistic effect of copper and low temperature over Listeria monocytogenes. Biometals 28(6):1087-1092.
  • Durack J, Ross T, Bowman JP. Characterisation of the transcriptomes of genetically diverse Listeria monocytogenes exposed to hyperosmotic and low temperature conditions reveal global stress-adaptation mechanisms. PLoS One. 2013.
  • Abdelhamed H, et al. 2015. A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes. Plasmid 81:1-8.
  • Hadjilouka A, et al. 2016. Expression of Listeria monocytogenes key virulence genes during growth in liquid medium, on rocket and melon at 4, 10 and 30 °C. Food Microbiol 2016, 55:7-15.

Project Title: Relation between Nutrition and development of Sleep Wake Cycle during the first 18 months

University of Michigan investigator(s) and unit:  Cristina Bares, PhD, Assistant Professor of Social Work

International colleague(s) name and unit: Drs. Patricio Peirano and Cecilia Algarin

Purpose: To establish if the features of the sleep wake cycle development is different according to feeding differences

 Rationale: Breast feeding is the gold standard for nutrition during the first year of life. However for several reasons (medical, socioeconomics, etc) there are infants that receive formula.

Sleep wake cycle development depends on adequate brain maturation. The goal of the study would be to establish if there are differences between a group of infant fed with breast milk vs a group that received formula.

Study design and methods:

From a population of 600 hundred newborns whose mothers accepted to participate in the study of growth and neurological development and nutrition at INTA, University of Chile, 150 were  recorded with actigraph ( a device that measure motor activity throughout the 24 hours) for sever days to determine the sleep wake cycle. The measure was performed at 4-6 months, 12 months and 18 months.

Anticipated undergraduate/graduate st1udent activities on project:

The collected data should be organize, analyze and compare among the different groups and at the 3 different ages.

Techniques/methods students should become familiar with in advance:

  1. Excel management
  2. Basic statistical knowledge.

Project Title: Intestinal colonization of different strains of Listeria monocytogenes and interaction with the gut microbiota

University of Michigan investigator(s) and unit:  Cristina Bares, PhD, Assistant Professor of Social Work

International colleague(s) name and unit: Paola Navarrete, PhD, Assistant Professor, Laboratory of Microbiology and Probiotics, INTA, University of Chile.

Purpose: To study the interaction of different strains of Listeria monocytogenes and the zebrafish (Danio rerio) model

Rationale: Listeria monocytogenes is a Gram positive foodborne bacterium that can infect a wide range of animals, including humans. The bacterium can produce septicemia, meningitis and chorioamnionitis which are associated with a high mortality. The severity of L. monocytogenes infection depends on the virulence of the strains and the immune status of the host. Immune-competent individuals that ingest L. monocytogenes contaminated food can have gastroenteritis that resolves in few days. However, immunocompromised individuals, infants, older people, and pregnant woman have a high risk to be infected and to have more severe symptoms.

The mechanisms involved in pathogenicity and virulence of this pathogen have been intensively studied. Some proteins and genes have been identified and related with the virulence of the pathogen. Some of these genes are involved with the adhesion and invasion of the pathogen into the host cells. However, to date, little is known about the intestinal phase of this pathogen, such as its replication in the gut or its relation with the gut microbiota.

A recent publication in a murine model showed that the gut microbiota markedly reduces L. monocytogenes colonization of the gut lumen and prevents systemic dissemination (Becattini et al., 2017). This study was performed with one pathogenic strain of L. monocytogenes (10403s). In our lab, we have a collection of L. monocytogenes isolates with different origin and phenotypic traits. In this project, we want to explore the intestinal behavior, such as growth or colonization, of these different strains of L. monocytogenes and evaluate if this behavior can be modulated by some early bacterial component of the gut microbiota of infant such as Lactobacillus, Bifidobacterium or Clostridium.

In this project we will use zebrafish. Compared to murine models, zebrafish has several advantages: they i) are easy to maintain, ii) develop rapidly (at 3 days post-fertilization (dpf) their mouth and digestive tract is open, so they can be colonized by different microorganisms), iii) in the early stages of development, larvae are transparent, which allows observing in vivo the colonization process, iv) the availability of transgenic lines expressing fluorescent proteins in different lineages of immune cells, allows the in vivo monitoring of physiological and pathophysiological processes, v) embryos and larvae are small enough to fit into 96-well plates, so it is possible to perform experiments with many individuals (high-throughput), vi) zebrafish’s genome has been completely sequenced, vii) methodologies to obtain germ-free fish or gnotobiotic have been described (and have already been standardized and implemented in our laboratory), viii) the digestive tract share extensive homology with mammalian and it includes the accessory organs such as the liver, gallbladder and pancreas. Additionally, the gastrointestinal tract also has a next-distal functional differentiation. The initial morphogenesis is completed at 3 days post fertilization (dpf), stage in which they can colonize the digestive tract with microorganisms (Bates et al., 2006; Rawls et al., 2004).

Study design and methods:

We will evaluate the effect of Lactobacillus, Bifidobacterium or Clostridium in the in vivo colonization of the zebrafish gut of different strains of Listeria monocytogenes. Colonization assays will be conducted according to methodology standardized in our lab (Caruffo et al, 2015 and 2016.). Colonization ability will be assessed from day 3 until day 9 dpf. These experiments will be independently performed 3 times.

Anticipated undergraduate/graduate st1udent activities on project:

The student will participate in the cultivation, DNA extraction, PCR amplification of different L. monocytogenes isolates as well as Lactobacillus, Bifidobacterium or Clostridium. and colonization studies in zebrafish larvae.

Techniques/methods students should become familiar with in advance:

1) Handling of bacterial cultures (aerobic and anaerobic with anaerobic jar)

2) Gram staining

3) DNA extraction from bacteria

4) PCR

Suggested readings (minimum of 3-5 articles):

Bates, J.M., et al. (2006). Dev Biol 297(2): 374-386.

Bates, J.M., et al. (2007). Cell Host Microbe 2(6): 371-382.

Becattini, S. et al. (2017) J. Experimental Med. 214(7): 1973-1989.

Caruffo M, et al. (2015). Front Microbiol. 7;6:1093. doi: 10.3389/fmicb.2015.01093.

Caruffo M, et al. (2016). Front Cell Infect Microbiol. http://dx.doi.org/10.3389/fcimb.2016.00127

Rawls, J.F., et al. (2006). Cell 127(2): 423-433.

Project Title: Intergenerational Effects of Iron Deficiency, Parental Obesity, and Drug use in Chile: A Pilot Study [ALERT **** Application deadline extended to November 15, 2017 for THIS PROJECT ONLY!****]

University of Michigan investigator(s) and unit: Jaclyn Goodrich, PhD, Research Assistant Professor of Environmental Health Sciences, School of Public Health and Cristina Bares, PhD, Assistant Professor of Social Work

International colleague(s) name and unit: Raquel Burrows, MD, and Paulina Correa, PhD, Public Health Nutrition Unit, INTA, University of Chile.

Purpose: The main purpose of the present study is to assess intergenerational effects of early life parental iron deficiency, obesity, and drug use. To do this, this pilot study considers the feasibility of recruiting children up to 2 years of age, born to young adults in the Santiago Longitudinal Study (SLS). SLS is a cohort of ~1,000 young adults who participated as infants in a preventative iron deficiency trial in Chile and have been followed ever since.

Rationale: Iron deficiency (ID) is the most common and widespread nutritional disorder in the world. As well as affecting many children and women in developing countries, it is the only nutrient deficiency which is also significantly prevalent in Industrialized Countries. ID during the fetal-neonatal period can cause acute and long-lasting modifications in physiological structures and/or functions leading to an increased risk of chronic disease and impaired cognitive functioning later in life. Additionally, evidence from human and animal studies suggest that ‘exposures’ to parental diet, weight status, obesity, drugs, environmental chemicals, and more can have long-lasting effects on the offspring and in some cases on multiple generations.

Epidemiological and animal data indicate that the risk of developing diseases, such as obesity and metabolic syndrome, is influenced by persistent adaptations to prenatal and postnatal environmental factors including nutritional deficiencies such as iron deficiency and socioeconomic circumstances. Moreover, the link between exposure in utero and disease development throughout life appears to involve epigenetic modifications. Epigenetic regulation consists of mitotically heritable yet reversible modifications (including DNA methylation) that influence expression of genes but do not change the underlying genetic sequence. Epigenetic modification is one mechanism by which parental health (e.g., obesity, metabolic syndrome, stress) and other exposures (e.g., ID, smoking, drug use, and socioeconomic status) during pregnancy and/or around the time of conception can impart lasting influences on a child’s development and health.

This study is being conducted in the context of an ongoing observational Chilean cohort of young adults who were enrolled as infants in a randomized controlled trial of iron supplementation between 1991-1996 in Santiago, Chile. Members of this group have been examined approximately every five years since their recruitment and are currently undergoing their fourth data collection cycle with a 22 year follow up. Many original study participants are now parents, and their children will be the focus of this study.

Due to iron’s role in brain reward circuits, we have substance use data for the SLS cohort. With the legalization of marijuana in Chile, we are interested to see any effects on the infants. Additionally, Chile has been hard hit by the obesity epidemic, making the epigenetics of energy metabolism another important research question. In this pilot sample, we aim to explore preliminary evidence for: a) intergenerational effects of early life ID on the offspring, b) adverse effects of parental obesity on the offspring, c) adverse effects of parental drug use on the offspring, and d) epigenetic modification as a mechanism linking these ‘exposures’ to offspring outcomes

Study design and methods:

  • Original Cohort
    • SLS participants have been part of the study since infancy to examine the behavioral and developmental effects of preventing ID. Participants were recruited at 4 months from public healthcare facilities in the southeast area of Santiago (n=1,791). All participants were enrolled as healthy, full-term infants weighing ≥3 kg at birth and from low- to mid-income families.
    • Of the 1657 infants who completed the preventative trial, approximately 1100 are still part of the study and have been followed with waves of data collection at 5, 10, 16 years and are currently being evaluated at 22 years
  • Pilot (G2) Cohort
    • A subgroup of original SLS members will be invited to participate in this pilot if they are pregnant or had a child ≤2 years of age. Partners are also invited to participate to collect information on father-mother-child triads. Additionally, participants are consented to access maternal and child medical records and neonatal dried blood spots (DBS).
    • Parental eating habits, physical activity, use of alcohol, tobacco and controlled substances, exposure to stress or adversity, and parenting practices are assessed via questionnaires.
    • Infants and toddlers are measured, weighed, and nutritionally assessed. A blood sample is collected to measure current iron status and to isolate DNA for epigenetics.

 Anticipated undergraduate/graduate student activities on project:

  • ​Coordinating with cohort’s contact/recruitment team to identify participants with very young children (<2 years of age).
  • Contacting potential participants: those who are parents of at least one child <2 years of age.  ​
  • Data entry, cleaning and updating of data files.
  • Administration of surveys to the parents during study visits and data collection from medical charts.
  • Data analyses

Techniques/methods students should become familiar with in advance:

  • Excel management
  • Knowledge of basic epidemiology concepts
  • Basic/intermediate statistical knowledge
  • Great interpersonal skills
  • Proficiency in Spanish

 Suggested readings (minimum of 3-5 articles):

Benyshek, Daniel C. “The “early life” origins of obesity‐related health disorders: New discoveries regarding the intergenerational transmission of developmentally programmed traits in the global cardiometabolic health crisis.” American journal of physical anthropology 152, no. S57 (2013): 79-93.

Burrows, Raquel, Paulina Correa-Burrows, Marcela Reyes, Estela Blanco, Cecilia Albala, and Sheila Gahagan. “High cardiometabolic risk in healthy Chilean adolescents: associations with anthropometric, biological and lifestyle factors.” Public health nutrition 19, no. 3 (2016): 486-493.

Carolan-Olah, Mary, Maria Duarte-Gardea, and Julia Lechuga. 2015. “A Critical Review: Early Life Nutrition and Prenatal Programming for Adult Disease.” Journal of Clinical Nursing 24 (23–24): 3716–29.

Faulk, Christopher, and Dana C. Dolinoy. “Timing is everything: the when and how of environmentally induced changes in the epigenome of animals.” Epigenetics 6, no. 7 (2011): 791-797.

Gahagan, Sheila, Sunkyung Yu, Niko Kaciroti, Marcela Castillo, and Betsy Lozoff. “Linear and ponderal growth trajectories in well-nourished, iron-sufficient infants are unimpaired by iron supplementation.” The Journal of nutrition 139, no. 11 (2009): 2106-2112.

Groom, A., H. R. Elliott, N. D. Embleton, and C. L. Relton. “Epigenetics and child health: basic principles.” Archives of disease in childhood (2010): archdischild165712.

Lozoff, Betsy, Marcela Castillo, Katy M. Clark, and Julia B. Smith. “Iron-fortified vs low-iron infant formula: developmental outcome at 10 years.” Archives of pediatrics & adolescent medicine 166, no. 3 (2012): 208-215.

Lozoff, Betsy, Niko Kaciroti, and Tomás Walter. “Iron deficiency in infancy: applying a physiologic framework for prediction.” The American journal of clinical nutrition 84, no. 6 (2006): 1412-1421.

Sen, Arko, Nicole Heredia, Marie-Claude Senut, Susan Land, Kurt Hollocher, Xiangyi Lu, Mary O. Dereski, and Douglas M. Ruden. “Multigenerational epigenetic inheritance in humans: DNA methylation changes associated with maternal exposure to lead can be transmitted to the grandchildren.” Scientific reports 5 (2015): 14466.

Szutorisz, Henrietta, and Yasmin L. Hurd. “High times for cannabis: Epigenetic imprint and its legacy on brain and behavior.” Neuroscience & Biobehavioral Reviews (2017).

Yohn, Nicole L., Marisa S. Bartolomei, and Julie A. Blendy. “Multigenerational and transgenerational inheritance of drug exposure: The effects of alcohol, opiates, cocaine, marijuana, and nicotine.” Progress in biophysics and molecular biology 118, no. 1 (2015): 21-33.