Lecture for: The Mediterranean Diet from an Italian Perspective “Let food be thy medicine and medicine be thy food” A knowledge and application-based continuing education activity for US pharmacists and dietitians Florence, Italy - November 6-9, 2018

1. DIET-MICROBIOTA
INTERACTIONS
THE MEDITERRANEAN DIET
AS A KEYTOA HEALTHY MICROBIOTA
Fabio Piccini MD PhD

2. • Doctor in Medicine
• MS Sport & Exercise Nutrition
• PhD Food Nutrition & Health
• Senior Consultant: Eating Disorders
and Obesity
• Co-founder & Director: Progetto
Microbioma Italiano
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3. DECLARATION OF INTEREST
The author reports no conflict of interest with respect to the content of this
presentation
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4. Learning objectives
At the conclusion of this lecture, you shall be able to:
• Define the human microbiome and the Mediterranean diet
• Discuss the effects of Western and Mediterranean diet on microbiota
and the links between microbial diversity and metabolic output
• Understand the epigenetic effects of the Mediterranean diet
• Describe the role of the Mediterranean diet in the prevention and
restoration of gut dysbiosis
• Summarize a Mediterranean food-shopping list
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5. By now you should know that…
• We are actually more non-human than human
• Trillions of microorganisms live all over our body and even inside
us
• These tiny microrganisms make up our MICROBIOTA
• Most of them (about 90%) live in the GI tract
• The combinations of these microbes, their genes, the
enviroment their are living and the stuff they produce is called
the HUMAN MICROBIOME
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6. A forced partnership
• The human microbiome can be considered a counterpart to the human genome.
• The genes in our microbiome outnumber the genes in our genome by about 100:1
• Our genome is fixed for life, but our microbiome changes over time and as we
grow up all our experiences influence the microbiome. Thus our microbial profile
changes along with us
• A few microbes cause diseases but most do not. In fact many are essential for
good health
• We are not alone.We aren’t just single individuals. We are ecosystems
• Disrupting our microbial ecosystems can cause disease and some diseases disrupt
our microbial ecosystems
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7. A word of caution is in order
• Image that your first image of an airplane was a picture of a debris field after a plane has
crashed
• Knowing nothing about aviation, you’d find it difficult to piece together what the airplane
looked like before the crash
• This analogy is akin to what researchers face when they try to understand how the human
microbiota works
• The vast majority of microbiota research has been performed on people from the USA or
Europe, the same individuals who are predisposed toWestern dieseases
• When we compare the microbiota of Westerners with diseases, to those without them, we
should be cognizant that the «healthy» group may not provide an accurate definition of a
healthy microbiota
• Since our definition of a healthy microbiota comes from studying Westerners, it’s likely
that our view of what is normal is higly distorted
7

8. Need more evidence?
• US immigration is associated with
loss of gut microbiome diversity
• US immigrants lose bacterial
enzymes associated with plant
fiber degradation
• Bacteroides strains displace
Prevotella strains according to time
spent in the USA
• Loss of diversity increases with
obesity and is compounded across
generations
8
Vangay et al., 2018, Cell 175, 962–972 November 1, 2018

9. Dietary choices shape the gut microbiota
• Our gut microbiota is shaped by what we eat
and drink
• Over the past four hundred yrs, the Industrial
Revolution brought unprecented and rapid
change to our diet
• If our bacteria were able to walk through
your average grocery store tasked with
finding something to eat they would face the
equivalent of humans trying to find food in a
Home Depot
• Thanks to our typical diet the average
Westerner’s gut bacteria are starving and
most of them belong on the endangered
species list
9
Image: https://hercanberra.com.au/

10. We are what our microbes eat
• We call it salad but most bacteria call it
home
• Organic labeled produce has different
microbial communities than the
conventionally grown food with the
organic microbes more diverse and more
probiotic in the organic veggies
• The fact of the matter is friendly bacteria
love fiber (not sugar, not salt, not trans
fats)
• Diets rich in fiber are associated with
higher microbial diversity, and a more
resilient microbiome
10
Image: Einladung Zum Essen, Pixabay Creative Commons CC0

11. Enter the Mediterranean Diet
11

12. In short
• Mediterranean diet doesn’t mean a lot of bread, pasta or pizza
• The actual diet can vary across different mediterranean countries yet there are
some core elements
• Among them are plenty of fruit and vegetables (often cultured), fish (both white
and oily), wholemeal cereals, beans and pulses, unsalted nuts and seeds, small
amounts of lean meat and low fat dairy (mostly in fermented forms),olive oil, fresh
herbs, and also a glass of red wine
• The Mediteranean diet is not only about food and drink
• Other traditional mediterranean lifestyle habits are part of its effect such as eating
slowly and with others, leading a more relaxed way of life, and doing regular
physical activity
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13. The Mediterranean diet pyramid
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14. Metabolic effects of the Mediterranean
diet
• The Mediterranean diet pattern is
increasingly associated with
improved metabolic health
• The modulation of microbiota and
the reduction of endotoxiemia are
two mechanisms that have been
proposed as responsible for such
effect
• Metabolic endotoxiemia (increase in
circulating levels of bacterial
endotoxins) has been proposed as a
cause of inflamation during
metabolic disfunction
14Guillermaut et al., Nutrients 9(11):1227 · November 2017

15. Mediterranean diet increase good gut
bacteria
• New research found that a Mediterranean
diet significantly increased the abundance
of «good» bacteria in the guts of monkeys,
compared to aWestern diet
• The researchers came to their conclusion by
studying a group of 20 monkeys. For 30
months, the monkeys were randomized to
one of two diets: a Mediterranean diet or a
Western diet
• The study showed that probiotic bacteria
(primarily Lactobacillus) were significantly
increased in the Mediterranean diet group
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16. From monkeys to men…
• In accordance with previous studies, a recent
spanish research shows associations
between the nutritional components of
traditional Mediterranean-style diets, certain
groups of beneficial bacteria, and
fermentable end products
• Moreover, studies have proven not only that
plant based diets are linked to an increase in
Bifidobacterium and Lactobacillus spp. but
also that they can stimulate the growth of
other beneficial bacterial species, especially
bacterial communities involved in butyrate
and methane production
16

17. MAC foods and gut health
• The gut microbiota of hunters-gatherers
and populations consuming a rural agrarian
diet is distinct and harbors a greater
diversity then the microbiota ofWesterners
• One possible explanation is the large
quantity of dietary fiber they consume
relative toWesterners
• Microbiota Accessible Carbohydrates
(MACs) serve as primary source of energy
for the distal gut microbiota
• Certain taxa decrease in abundance upon
reduced MACs and are not effectively
transferred to the next generation
• Such a change is magnified over
generations
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18. Consequences of a Diet Deficient in MACs
• In the first scenario, a high-MAC diet that has
few simple sugars, the major contribution of
carbohydrates to host metabolism is in the form
of the SCFA fermentation end-products of the
microbiota
• In the second scenario, the low-MAC Western
diet results not only in a loss of beneficial
microbial metabolites such as SCFA, but also in
selection of a distinct microbiota that may seem
foreign to the host. Increased representation of
mucus-utilizing microbes, decreased gut
motility, and increased calories in the form of
sugar and fat may synergize to cause Western
diseases
• MACs include carbohydrates from diet, host
secretion (e.g., mucin glycans), or other resident
microbes that serve as a metabolic input for
members of the microbiota.
Sonnenburg. J. Cell Metab. 2014 Nov 4; 20(5): 779–786. 18

19. Diversity matters
In an ecosystem like that of the gut , diversity can be a buffer against system collapse.
This is an example of very low gut bacterial diversity from a gut microbiome sequencing
obtained from an anorexic patient
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BMR Genomics NGS sequencing for Progetto Microbioma Italiano

20. Low diversity means dysbiosis
BMR Genomics NGS sequencing for Progetto Microbioma Italiano
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21. Dysbiosis means troubles
• There is growing evidence that
dysbiosis of the gut microbiota is
associated with the pathogenesis of
both intestinal and extra-intestinal
disorders
• Intestinal disorders include
inflammatory bowel disease, irritable
bowel syndrome (IBS), and coeliac
disease
• Extra-intestinal disorders include
allergy, asthma, metabolic syndrome,
cardiovascular disease, and obesityAdapted from: Piccini F. Microbioma Intestino E Salute, LSWR

22. Can we recover?
• How important is the loss of
coevolved microbial species versus
the loss of microbial metabolites?
• Would a more sensible diet that
increases MAC content be sufficient
to recover microbiota diversity, or
have species been irreversibly lost?
• Does imposing a high-MAC diet on a
Western microbiota present new
incompatibilities and potential
unanticipated negative health
consequences?
Sonnenburg. J. Cell Metab. 2014 Nov 4; 20(5): 779–786.
22

23. How much is lost?
• An independent study of rural
groups in Malawi and Venezuela
revealed greater bacterial
diversity and stark differences in
microbiota composition of non
modern societies relative to
Western groups
• Perhaps their most significant
finding is the much larger extent
of bacterial diversity in the
foraging microbiota compared to
the industrialized group
Sonnenburg. J. Cell Metab. 2014 Nov 4; 20(5): 779–786.
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24. Our pre-agricultural ancestors
• The Hadza are one of the last remaining hunter-
gatherer tribes in the world
• Their diet changes during seasons; they consume
mostly meat and vegetables in the dry season
and mostly fruits and vegetables during the wet
season
• Their microbiome changes drastically with the
seasons
• Certain species of microbe disappear entirely
from the gut as the season changes and reappear
again when that season comes back
• They seem to periodically lose the microbes we
ourselves have lost forever
• Hadzas consume on average 100-150 grams of
fiber a day, Americans about 10-15 grams,
Mediterranean populations about 25-35
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25. Re-thinking the inulin solution
• Dietary soluble fibers are fermented by gut bacteria
into short-chain fatty acids (SCFA), which are
considered broadly health-promoting. Accordingly,
consumption of such fibers ameliorates metabolic
syndrome
• However, incorporating soluble fiber inulin, but not
insoluble fiber, into a High Fat diet, induced icteric
hepatocellular carcinoma (HCC) in mice
• Such HCC was microbiota-dependent and observed
in multiple strains of dysbiotic mice but not in germ-
free nor antibiotics-treated mice
• Thus, its benefits notwithstanding, enrichment of
foods with fermentable fiber should be approached
with great caution as it may increase the risk of HCC
25

26. The Mediterranean solution
• The Mediterranean-style diet was first made famous by researcher Ancel Keys,
who in the 1950s launched the Seven Countries Study, examining the relationship
between diets in various countries and their prevalence of heart disease. Both
Greece and Italy made the list of the lowest instances
• Keys has since been criticized for his flawed study techniques, however, he helped
to bring attention to the Mediterranean diet pattern that has consistently helped
to stave off disease
• The Mediterranean diet differs from MyPlate, the USDA's dietary guide, in that it’s
not about measuring serving sizes – rather, it’s about eating high-quality, nutrient-
dense, fiber-rich foods
• The life-extending powers of the Mediterranean diet have withstood the test of
time – with one of the world's highest percentages of centenarians to prove it
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27. What the Sicani mountains
centenarians eat
• In the Sicani Mountain zone there are
more centenarians with respect to the
Italian average
• Centenarians living in this area tend to be
physically active, non-obese, small in
stature, with a regular BMI (23.6 ± 3.1)
• All the centenarians live in a family home,
usually with their relatives
• The Mini Nutritional Assessment showed
a high adherence to the Mediterranean
nutritional profile (no white bread, low
amount of pasta, no sweeteners or sweet
beverages, no canned foods or industrial
foods, no cookies, pastries or snacks)
Vasto e coll. Immun Ageing. 2012; 9: 10.
27

28. The Sardinian centenarians lifestyle
• They are largely sheperds which
occasions regular, low intensity
physical activity
• Their diet is mostly plant based, plus
some whole wheat bread, some
cheese made from grass fed sheep
and a wine that has 3 times the level
of polyphenols than any known wine
in the world (the Cannonau)
• They have organized their society in a
way that the older you get, the more
equity you have, the more wisdom
you are celebrated for
28Image: Jarmoluk, Pixabay Creative Commons CC0

29. Genetics vs. lifestyle
• A Danish twin study established that only
about 15-25 percent of how long the
average person lives (within certain
biological limits) is dictated by our genes
• The other 75-85 percent is dictated by
our lifestyle. This means: if we can find
the optimal lifestyle of longevity we can
come up with a de facto formula for
longevity
• Recently, epigenetic studies have shown
that epigenetic modifications, modulated
by both genetic background and lifestyle,
are very sensitive to the aging process
and can either be a biomarker of the
quality of aging or influence the rate and
the quality of aging
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30. What is epigenetics
30

31. Diet, epigenetics and the centenarian
gut microbiota
• Centenarians represent the best model of “successful” aging showing a lower incidence of
chronic illness, a reduction of morbidity and an extension of health span in comparison to
octogenarians and nonagenarians from the same cohort
• When compared to other elderly people, centenarians stand out as a separate population,
their gut microbiota showing higher diversity in terms of species composition
• Several butyrate producers are found in lower amounts, while others increase suggesting
the existence of bacteria characteristic of longevity
• The microbiota of centenarians is enriched in facultative anaerobe bacteria mostly
belonging to Proteobacteria which have been recently redefined as “pathobionts”
• In extreme longevity, this change is counterbalanced by an increase in longevity-adapted
and possibly health-promoting subdominant species (e.g., Akkermansia, Bifidobacterium,
Christensenellaceae)
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32. The NU-Age Project
• The NU-AGE rationale was that a one year Mediterranean diet newly designed to meet the
nutritional needs of the elderly, would reduce inflammaging in fully characterized subjects
aged 65-79 year of age, and would have systemic beneficial effects on health status
• Volunteers partaking in the study were divided into two groups, a control group,
consuming their normal diets and an intervention group, eating a Mediterranean style diet
rich in fruits, vegetables, fish, low-fat meat, nuts and olive oil.
• Before and after the dietary intervention a comprehensive set of analyses, including omics
(transcriptomics, epigenetics, metabolomics, metagenomics) have been performed to
identify the underpinning molecular mechanisms of aging
• After 5 years, the EU-funded project, NU-AGE came to an end, bringing new dietary
guidelines for over 65-year-olds. By comparing the two groups, this study allowed
researchers to determine the effect of the diet on various markers related to health
32

33. Nu-Age Project results
33http://www.nu-age.eu/Upload/ReadmoreDocuments/48/Nu-AgeLeaflet_FINAL.pdf

34. Kids weaning: a good place to start
• Weaning is an opportunity for long term microbiota health, since is one of the most
dramatic rearrangement periods for the microbiota in a person’s life
• Thus it makes sense to wean children onto food that will maximize the diversity of their
microbiota
• We started our daughter Gaia on solid foods the way many Italian parents do, first
introducing vegetables pureed (peas, carrots, broccoli, etc.) then moving to fruits
• This order of vegetables-before-fruits followed the reasoning that if babies have too much
fruits first, they won’t like the the less sweet vegetables
• Along with veggies and fruits we fed our daughter with rice cereal, oatmeal, along with
other typer of grain cereals, dairy products and fish
• As she got older we introduced portions of our own food since we wanted to develop a tast
for real food; the food that hopefully she should consume for the rest of her life
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35. Gaia’s microbiome 23 yrs after weaning
Ubiome Gut Explorer NGS sequencing.Tested on 09/20/2018
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36. Kids nutrition: tricks of the trade
• Of course, feeding your children what
you eat, only makes sense if you eat a
healthy diet yourserlf
• Eating for health and a robust microbiota
needs to be a family endeavor
• Since persuading a toddler to eat
steamed vegetables or fighting with her
at the dinner table may not be easy, I’d
rather suggest to use mealtimes as an
opportunity to inform your children
about the benefits of feeding they
microscopic pets
• Bat is the name of a bacillus who teach
kids the importance of microbes for the
planet’s ecosystems as well as for human
survival and food production
36Blasco Pisapia. Cover Design for: Il Mondo Di Bat, LSWR in press

37. Perfecting the Mediterranean Diet with
fermented foods
• Traditional fermented foods
constitute a significant part of the
Mediterranean diet and represent a
gastronomical heritage that needs to
be preserved and protected
• The manufacture and sensorial
properties of such products relies on
complex and poorly defined microbial
consortia activities
• In this context, microbiota plays a
central role in products manufacturing
and is responsible for their beneficial
health benefits
37
http://www.return2health.net/articles/fermented-foods-basics/

38. Mediterranean fermented foods
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• Fermented milk products are dairy foods that have
been fermented with lactic acid bacteria. Many
different types of cultured milk products can be found
around the Mediterranean countries
• Fermented vegetables have a long history in
Mediterranean culture. The Roman writers Cato (De
Agri Cultura) and Columella (De Re Rustica) mentioned
preserving cabbages and turnips with salt. Cultured
veggies increase the bioavailability of nutrients and If
unpasteurized and uncooked, also contains live
lactobacilli and their byproducts like enzymes,
bacteriocins, etc. which can improve digestion and
promote biodiversity in the gut flora
• WARNING: Individuals that have a histamine
intolerance, caused by a decreased activity of DAO
enzyme, may not be able to tolerate fermented foods
Image: Fabio Piccini MD PhD

39. How to make your diet more
Mediterranean
You can make you diet more mediterranean-style by:
• eating plenty of wholemeal cereals, beans and pulses
• eating plenty of fruit and vegetables
• including fish in your diet
• eating less meat
• choosing products made from vegetable and plant oils, such as olive oil, vinegar oil, flaxseed oil,
etc.
• choosing local, seasonal, eco-friendly foods
• learning food fermentation
• reducing sugar and salt
• restricting calories and/or skipping meals at times
39

40. Take home message
• The Mediterranean diet it is as straight
forward as it is un-American
• It’s low in red meat, low in saturated fats,
low in salt and sugar, low in hyper-
processed carbs
• It’s high in just about everything else.
Healthy fats, vegetables, fruits, fish, eggs,
fermented diaries and cultured foods. And
there is also good wine
• Will restore and improve gut bacteria by
adding diversity and increasing probiotic
strains
• Give it a try then spread the word to your
patients
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Image: Laura Borowski, Pixabay Creative Commons CC0

41. Thank you for attention
f.piccini@univpm.it
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