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I would like to thank Ozolea for giving me the opportunity to clarify an interesting yet complicated topic. Also this morning at a conference there were people operating in the field but with scares scientific knowledge, where infective and contagious diseases were confused and the term infectious was used as a synonym of contagious. These are two totally distinct concepts.

Even when we talk about antibiotic resistance: this is a term that needs to be understood. We are often dazed by incorrect information. Antibiotics have been discovered by a great researcher, a scientist that I like to call a genius: Alexander Fleming in 1928 returns to his lab after a few days of absence and on a slab where the replicated some bacteria he founds a mould, and he notices that around that mould bacteria are not growing. Normally, a researcher would have discarded the slab and replaced with another one. The genius, instead, understood what happened: in that mould there were some active elements that somehow inhibit the grow of bacteria. This has been the first description of antibiotics, that is substances that bacteria and micro-organisms in general use to compete among each other. Micro-organisms have been the first to appear on earth and of course they had to colonize and compete for the territory, environment and nutrition.

These are antibiotics: something produced by bacteria and microorganisms in general in order to create their own ecologic niche and predominate on others.

Nowadays, we call antibiotics some small packages we buy at the pharmacy. In reality, since the very beginning both humans and animals activated defense mechanisms to fight the presence of pathogenic micro-organisms. The immune system is one of these, but not the only one. There is a specific, non specific, and action based immune system that allows the subject, both human and animal, to fight the presence of pathogenic micro-organisms. Today we often hear talking about microbiome: a microbiome is a system of bacteria that colonize a specific area. These bacteria are crucial for the animal welfare: we don’t have to eliminate them all, we need to eliminate those that create problems. Because we do need the others, they are useful as they compete with pathogens producing antimicrobial substances that fight pathogens growth. Therefore you understand these concepts are quite complicated: biology is never black or white, but is made of colors that often blend and create very complex contexts.

Sometimes the immune system is not sufficient by itself to keep this delicate situation under control, and that’s when an animal or a human get sick: at this point, with Fleming’s discovery we have at our disposal medicines that contain this active ingredients, which are active towards other micro-organisms.

Then we can use them in treatment. This is the project: having a medicine that allows me to eliminate a microorganism that creates an alteration, that causes a tissue damage, that provokes a disease. This is the path.

The concept of antimicrobial, instead, is a little wider: we often hear talking about antibiotic and antimicrobial as synonyms. An antimicrobial is a natural substance, that can also be synthetic or semi-synthetic, that kills a bacteria and inhibits its growth. When this can be well tolerated and have a selective toxicity, then it becomes an antibiotic, that can be used in treatment. A disinfectant is a antimicrobial, an antibiotic instead is something that we use in treatment: these are two concepts similar in certain ways but with important differences.

Selective toxicity is the ability an antibiotic has to have a higher or exclusive toxicity towards pathogens, and at the same time not to cause any problem, or a minor and less invasive problem, in the host organism.

Selective toxicity is due to a series of different mechanisms: antibiotics take advantage of the different characteristics given by the fact the bacteria are prokaryotes and the animal or human organisms are eukaryotic. In fact eukaryotic cells dispose of action sites that the antibiotic cannot reach, have antibiotic elimination systems that bacteria don’t have, and display of a series of mechanisms that allow define this type of toxicity as “selective”.

Another important concept is that antibiotics don’t have any power against viruses because in viruses there isn’t any target organ: it must be very clear that antibiotics perform their action within the competition between micro-organisms but they are inactive towards viruses. We’ll see that in viruses there are other mechanisms that we will get to study.

So, when we decide to use an antibiotic we must make sure that we are fighting a bacterial action, because if the infection is viral we are creating an additional problem to the environment, to the animal and to the system. The first thing to do before taking an antibiotic is performing an accurate diagnosis: here the professional, the veterinarian, the doctor play an important role, having to provide an extremely accurate diagnosis. This is the first aspect of antibiotic resistance: in order not to use antibiotics in vain, uselessly, in order not to waste money, and not to uselessly discard antibiotics in the environment, we need to perform an effective diagnosis, based on the principle that if I have a bacterial disease I use an antibiotic, and if I have a viral disease I will use something else.

The introduction of antibiotics in human and veterinarian medicine had a series of positive effects, extremely important: in the farm it guarantees the quality and wholesomeness of products, animal welfare, reduces the spreading of pathogens, has a series of very important positive effects. The improper use and sometimes the excessive use can be for example for a viral infection, where it has no effect, as well when I use it in an excessive dose, or not in the correct way, that is without observing the withdrawal times, or without observing those conditions that researchers recommend with respect to the procedures of correct use of antibiotics.

If I do all this, where does it lead?

It leads to a mechanism of antibiotic resistance.

This because from a genetic point of view bacteria are plastic organisms: being the first organisms appeared on earth, they are able to resist to any condition.

They are extremely efficient organisms, so that if I use an antibiotic they are able to activate antibiotic resistance mechanisms, that is mechanisms that allow bacteria to resist the antimicrobial activity.

The problem of excessive and improper use of the existing antibiotics is of course nowadays an important problem, also because human-animal-environment cannot be seen anymore as independent systems but they do interact: humans, animals and environment share the same micro-organisms in the same environment.

The problem of antibiotic resistance and antimicrobial resistance is an actual problem, by now widespread and perceived worldwide: it’s a time bomb. We need to be very careful and take preventive measures: at an institutional level this is already in process.

What is antibiotic resistance: bacteria are extremely plastic micro-organisms. When I use an antibiotic with the correct dosage, bacteria activate defense mechanisms that make them more resistant to higher antibiotic doses.

Their capacity to reproduce, to survive to the presence of antibiotic is a natural phenomenon that however need to be checked and studied.

How does antibiotic resistance technically occur?

Some micro-organisms developed by themselves mechanisms that block the effectiveness of an antibiotic: they are able to activate efflux mechanisms (they throw it out), don’t let it in, they have antibiotic resistance mechanism of their own. Bacteria are not static, their genome is very plastic and is able to substantially change, to recombine, to acquire genetic material from the outside, this made possible that they acquire certain characteristics and they can transfer them among each other. When a micro-organism is resistant it can transfer the resistance to another micro-organism that is present in the same environment, and it can do this even if it is dead: the genome of the killed bacteria can circulate in the environment or can circulate within other microbic species. In this way they become “acquired resistances” and then “multiple resistances”: a bacteria that directly became resistant to an antibiotic can then become resistant to another antibiotic, for being entered into contact with a genome that was resistant to other bacteria, making the problem bigger and bigger.

However the antibiotic resistance is not a phenomenon that followed the discovery of antibiotics: 30.000 years ago, from a publication of Nature of 2011, permafrost bacteria from 30.00 years ago have been found resistant to penicillin and tetracycline. Therefore you understand that antibiotic resistance is a natural phenomenon: even in our intestine there are bacteria that in order to compete among themselves carry out antibiotic resistance mechanisms that are transmitted through different mechanisms. There are some phages, bacteria and viruses that hit a bacterium and transport its genes to another through complex mechanisms: in our intestine we have an extra logarithm of bacterial cells compared to the cells we are made of (bacterial cells are 10 times the number of cells that our organism is made of). Therefore we carry around a number of pals: in a grain of feces there are more that 100 billions of bacteria and when you take some probiotics you intake 4 billions (kind of useless).

Antibiotic resistance is a mechanism used by bacteria since forever.

Why do we realize now about the antibiotic crisis? Because since their discovery every 2-3 years we discovered a different type of antibiotic, therefore if bacteria became resistant to an antibiotic this was quickly replaced with a new molecule: for a certain number of years antibiotics represented an important tool for doctors, veterinarians and for all healthcare providers. It was an obstacle race where the discovery of new molecules guaranteed to control the phenomenon.

Today, since approximately 15 years, we don’t find new molecules. The problem arose and we started to think about it, considering that these thoughts must keep into account also a productive system based on ethics, more respectful towards the environment, individuals, the productive system, the consumers themselves: therefore it’s a wider and more interesting project and at the same time, helpful from a health point of view. Because we have this problem: since a decade we can’t find new antibiotics, while bacteria are continuing to evolve in their antibiotic resistance.

What normally is not said is that it is true that bacteria activate new antibiotic resistance mechanisms, but if they are not submitted to the selective pressure of some antibiotics, they become sensitive to old antibiotics. We performed a number of researches in the Neaples area with maximalist doctors in urinary tract infections there is an important resistance to fluoroquinolones widely used but these bacteria return to be sensitive to furanes and nitrofurantoin, that is to old antibiotics. To create shared paths of selective use of antibiotics could be a strategy: for the bacterium activating mechanisms of antibiotic resistance is a job that not even bacteria do for nothing, moreover for bacteria that always work in the maximum efficiency, maximum results with the minimum effort. As soon as they lose the necessity to resist to an antibiotic molecule because that molecule has not been used for a while, they also lose the resistance to that molecule and return to be sensitive. In this way it could be useful to create new therapeutic treatments.

In the detail, how does antibiotic resistance occur?

In a population of bacteria, at a certain point we treat with antibiotic and we kill all the bacteria, except for one, that is antibiotic resistant. This resistant replicates (bacteria have a very violent and quick replicative system: every 20 minutes some bacteria can duplicate and that’s how the resistant strain is selected): at a certain point we will have a population entirely resistant. Then we need to increase the antibiotic concentration, but it’s a game we cannot carry on forever. On the other hand we saw that often these bacteria become sensitive again to other antibiotics, and this could represent a strategy: it is understandable that the guideline remains a reduced, targeted and effective use.

Antibiotic resistance is a global issue: in the last 40 years the presence of antibiotics drastically reduces infection caused mortalities, but the risk is that in 2050 we may return to a pre-antibiotic era. This fear spread worldwide lead politics to insist for a reduced and more efficient use of antibiotics, to the extent that in the national chain production starting from January 1, 2019 the use of the electronic prescription will be mandatory. This will permit an accurate control of the use of antibiotics and will allow use to improve and optimize the use of antibiotic. It is certainly an extra effort but it’s meant to create a chain production that is ethic and sustainable: even if it may cause some problem, this is the step we need to take in order to preserve our future generations.

The other important notion is prevention: when we use an antibiotic it’s because we lost, because in some way we did something wrong. Then what we need to do is to bite the time: the soccer player that bites the time usually takes the ball away and this is what we need to do. To work towards prevention, that is to work in a number of situations that allow us to prevent: if the animal is in perfect shape, it’s hard it will get sick, it just doesn’t. If you look at our generation, it has an expectation of life of 80 years, and has a considerable lower number of infections and serious diseases compared to the population living 100 years ago, or to populations exposed to wars, malnutrition, other problems that affect the welfare. It is the same for animals: we need to work keeping in mind to improve the animal management, also through mechanisms of biosafety, improvement of hygienic and sanitary conditions, nourishment, as well as vaccination programs: overall the animal welfare. If you go in an overcrowded room in which you are thigh and bottled up, you feel bad, and when you get out you have a cold and your eyes weep: for the cow it happens the same. The more the animal feels good, the more it succeeds in fighting antibiotic resistance: this is a perspective of less use of antibiotics.

There are then some treatments that are effective and represent an alternative or a prevention of the use of antibiotics: I use them because it’s best not having to have to use antibiotics. Then when the animal gets sick I have to use the antibiotic, and I have to use in a correct and responsible way. Among these systems we find new vaccines, immunostimulants, bacteriophages, antimicrobial peptide, pre and pro symbiotics, new plant derived products, ozonized oils for example, or quorum sensing or biofilm inhibitors.

What is the quorum sensing? Bacteria don’t work each on its own, they talk to each other, they send messages among each other when they need to create a biofilm or harm the tissue and they also send messages to block their replication. It is extremely important to study these mechanisms. Also using bacterial competition can work: we saw that if I have a certain type of lactococcus in the curd, this competes against listeria, which is monocytogenes and in order to replicate and become a problem must be present in higher concentrations in lactoccoccus are present, as they reduce its virulence.

Other mechanisms can be for example the replacement of antimicrobial principles with substances that don’t originate residue, therefore it’s something present in the environment and does not cause any problem. Ozone is a very instable molecule that transforms almost immediately in oxygen, not creating any problem to the environment. Of course it needs to be used in the correct way.

The research we are carrying on in collaboration with Ozolea is to verify the study of the resistome. What is the resistome? First of all the resistome is a study that uses a different research philosophy based on proteomic. Proteomic is the study of proteins that are expressed by a series of genes, that is a genome. The genome is something statical that tells me who they are. It’s like going to the vital record office and get the vital record register: I see the names of all the presents but I don’t get information on what they are doing in this moment. Proteomics reveals if these bacteria are doing something, if they retired or are up to something else.

This morning in a conference it was presented a research by Altroconsumo that examined pieced of chicken breast and found genes of antibiotic resistance, and we are lucky you found them, it’s a totally normal phenomenon on earth: having genes of antibiotic resistance it means that they were there, we cannot know if bacteria with a certain resistance are still there, if they are actively doing something or if they left. We know that some bacteria with antibiotic resistance genes were there, but the research doesn’t tell anything more, and that’s normal. If we consider the bacteria present in our intestine we’ll find tons of antibiotic resistance genes, but I don’t think that we are all currently under antibiotic treatment. Therefore they are two distinct notions: the fact that some genes are present or the fact that there are some proteins linked to the gene. When the gene is expressed, I have the protein. This is proteomics: proteomics searches for the expressed protein. I won’t search only of genes of antibiotic resistance any more, but I investigate if these genes are doing something, if they are performing any action or already retired.

What we saw in this research not yet entirely published for example is that with proteomics we can study those proteins linked to antibiotic resistance genes in a bacterium. I cited just a couple, two paths therefore two proteins liked to two paths of antibiotic resistance. So if we find those proteins that means that that antibiotic resistance bacterium is present. This only from a bacterium. But we are more complicated organism, we don’t have just a bacterium but a ton. In any biological context interactions are complex: there are different bacteria that proceed from humans, animals, environment, therefore it’s an extremely complex environment. And here the term “resistome” is forged.

The resistome is something that takes a picture of those protein linked to resistant genes: the presence of proteins means that those resistance genes are expressed, are working, there is someone who is working for the antibiotic resistance.

What we are doing also with Ozolea is to check if these antibiotic genes can be detectable in the milk and if we can distinguish between resistance genes in farms that use antibiotics and in farms that don’t use them. This is what we did: we checked the antibiotic resistance genes expression in the microbiome, that is among all the bacteria present in the milk. Talking about proteins, therefore expressed genes, not just genes that are present but genes that are actually working, is a totally different thing than checking if the gene is there, that doesn’t mean anything.

I will spare you the method: these are very complex procedures that need powerful database and it takes days to interpret very complex data. What we saw, data not yet published, is that in the milk there is a series of peptides, approximately 8000, 5724 of which belongs to bacteria. I spared you the methodological part: in effects from the milk we were able to select only bacterial cells, from which we evaluated all the proteins that were present, and of these selected proteins, 6000 out of 8100 were specifically belonging to bacteria. 952 of these were linked to antibiotic resistance genes, therefore we found 952 proteins related to antibiotic resistance genes.

I spare you the frequency of proteins involved and the different mechanisms of antibiotic resistance.

With Ozolea we selected two farms in which we applied the system described above: a farms that regularly uses antibiotics, and another that opts for a prevention approach and therefore uses much less antibiotics, if none. What we noticed is that in two similar farms, that use the same antibiotics for therapy, a sample of milk has been collected: in the sample collected from the farm that chose a prevention approach we didn’t find proteins linked to antibiotic resistance. Consider that is a farm: I received this morning data from another farm that uses Ozolea that instead expressed some antibiotic resistance genes, but liked to bacteria or antibiotics not used in the treatment. These are data that we are currently processing, for which some time is needed. Therefore in this case there weren’t antibiotic resistance proteins because probably it was a while that antibiotics weren’t used. In the other control farm instead where antibiotics are regularly used also for dry cows, as you can see we found 13 antibiotic resistance related proteins, therefore there were 13 signals of bacteria which were activating antibiotic resistances: some are due to the environment, that means they are bacteria that activate these genes because they need to compete among each other, but there was also Coli or Pseudomonas which are instead pathogen bacteria, potentially pathogens. So it is clear that the antibiotic resistances we found are all related to beta lactamases, therefore penicillins and cefalosporins. We will go back and recheck to see if in this farm these types of antibiotics are actually used, but it is very likely so. This extremely interesting path allows us to understand and to define a system a little more sophisticated, a little more efficient compared to the genomic study that only gives an idea of the presence of genes.

In conclusion, if we use an approach that allows us to improve the farm and the animal performance, it is clear that we reduce the pathologic conditions that require the use of antibiotics. Obviously through a targeted use and only in pathological conditions and for the correct period of time, we avoid antibiotic resistance and we incur in fewer costs. Everything ends with a sustainable and ethic production.