Scientific Dissemination

Lucky accidents and the discovery of the first antibiotic

Part of the history of science has always stated that Alexander Fleming, the Scottish physician, working at St Mary´s Hospital in Paddington, London, accidentally discovered one of the greatest pillars of modern medicine. An event so significant that it would change our health forever: the discovery of antibiotics. We know these drugs as a group of drugs that kill bacteria. But, they are more than that, they are diverse and can be classified according to their origin as natural, semi-synthetic or synthetic. If they prevent bacterial growth without destroying the bacteria they are called bacteriostatic, if it destroys (killing) them it is known as bacteriolytic (where most of them are grouped). They can also be grouped by their mechanism of action, or by their chemical structure (Table 1). For example, if we take the 100 most common antibiotics today, we could group them into about 16 different families. On the other hand, if we consider the number of microorganisms on which they apply their effect, there are broad-spectrum, medium-spectrum, and short or specific spectrum antibiotics. If we consider their duration in the body, some take weeks to be disposed of and others only a few hours. This count could be overwhelming, so let's start from the beginning (^{Goodman et al., 2008}).

Penicillins and antibiotics in general usually have a first, second and even third line prescription, but the choice is determined by the bacteria rather than by the structural component of the antibiotic. It is possible that a drug is the first choice in some infections and the second or is ruled out in others. Few fourth-generation penicillins are available outside of Piperacillin, due to widespread resistance to beta-lactamases

Initially isolated from the fungus Penicillium notatum, 6-aminopenicillanic acid reached mass production from the method developed by Ernst Boris Chain and Howard Walter Foley, who, together with Alexander Fleming, would be awarded the Nobel Prize in Medicine in 1945. The effectiveness of penicillin as an antibiotic allowed its frequent use in clinical practice and its demand increased during the twenty years after its discovery (^{Lobanovska &Pilla, 2017}). A cure had been found for dozens of infectious diseases simultaneously. In the context of the Second World War, the wounds inflicted on soldiers were no longer lethal. Hundreds of lives around the world were saved daily, thanks to basic research and pharmaceutical investment in these projects. As a result of this synergy, synthetic penicillins with oral routes of administration (the most easily accessible) such as penicillin V, ampicillin, flucloxacillin, cloxacillin and amoxicillin appeared shortly afterwards, thus inaugurating the era of antibiotics.

Alexander Fleming was not known for his immaculate order within the laboratory. In fact, he had gone on vacation to Scotland, leaving several culture plates on the workbench. Upon his return, on the morning of Friday, September 28, 1928, he began to discard them. It had been about a month since he had performed those bacterial cultures, and they had all been contaminated by a mold-like fungus that grows on damp bread. They were beyond useless! In the first instance, he put some of these plates in a basket to throw them away, but was interrupted by a colleague, then when he showed him his work in the cultures, he realized that the fungus that had grown inside the plate had formed a halo of inhibition in the bacterial culture ... in other words, the fungus secreted something that had destroyed all the bacteria around it. This observation was what led him to isolate the microorganism and look for this antibiotic substance in it ... the rest, as they say, is history, which ninety years later begins to be rewritten, since many bacteria silently began an adaptation process that allowed them to survive and grow in the presence of these first antibiotics. This innocent antimicrobial resistance (AMR), today constitutes one of the greatest threats that threatens the current and future world population: infection by pathogenic bacteria with AMR or also called superbugs, which are resistant to most antibiotics (^{WHO, 2020}) ‎.

While penicillin was the first antibiotic to be developed, it was not the only one known at the time. The bactericidal activity of lysozyme (a protein with the ability to break down other proteins) was already well known, and which by the way was also discovered by Alexander Fleming in 1922, curiously after his nasal fluids reached a culture of bacteria. One day, he was checking a culture plate, and then accidentally sneezed at it ... but he did not throw it! And this is perhaps what made Fleming a great researcher, not his disorder, but his curiosity and great observation skills. This explains why years later when he saw the fungus growing in those agar plates full of bacteria, he would not throw them right away. His experience with lysozyme had made her reasonably curious to know what was going on in those culture plates.

Years later, lysozyme was discovered to be part of the defense systems of many animals. It is very efficient at keeping bacteria away and therefore is secreted in tears and saliva, however, because its way of action and efficiency are less effective than that of penicillin (^{Fortoul, 2019}).

Colors under the microscope: first identification tool

Antibiotics such as penicillin act very fast, but to talk about their mechanism we must first talk about bacteria. We can differentiate the millions of bacteria into two large groups: Gram-positive (+) and Gram-negative (-) bacteria. This name is due to Han Christian Gram, who made a blue-violet dye, which penetrated some bacteria, staining them a blue-violet color. So he could easily classify them as positive, those bacteria that stained, and negative those that did not. The key to this was the bacterial envelope, since the negative or Gram (-) had what is known as a cell wall, a rigid protective envelope that does not allow the passage of the dye. In this group, we can find many of the bacteria that make us sick, such as Neisseria gonorrhoeae, which causes gonorrhea - a sexually transmitted disease (STD), Neisseria meningitidis that causes meningitis or Moraxella catarrhalis, whose infection causes respiratory symptoms, among others. Some of them mainly cause respiratory diseases (Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), urinary diseases (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens) and gastrointestinal diseases such as Helicobacter pylori and Salmonella. Other members of this group are opportunists and attack when your body is weak, which is why they are associated with nosocomial infections such as Acinetobacter baumanii.

On the other hand, positive or Gram (+) bacteria do not have a protective covering like the cell wall, instead, they have a pair of semi-permeable membranes that provides protection from the external environment, so their membrane allows the passage of the dye and they stain blue-violet when viewed under the microscope. This group includes both mobile (via flagella) and immobile species with bacillus (Bacillus, Clostridium, Corynebacterium, Lactobacillus, Listeria) or coconut (Staphylococcus, Streptococcus) shape (^{Whitman et al., 2012}).

The bacterial counterattack: the first resistance

The cell wall is an essential component of bacterial protection that is mainly composed of sugar chains alternating with other molecules. The antibiotic activity of penicillin is conferred by the ability to block cell wall synthesis in Gram negative bacteria. When this drug reaches the place where the wall is made, it binds to the protein responsible for placing it on the wall (that is, it sticks to an enzyme, but can no longer be detached), therefore, it disables the enzyme that makes the cell wall.

Penicillin mimics the structure of the last two D-alanine residues present in all these peptides, in such a way that the transpeptidase enzyme binds covalently to penicillin and is irreversibly blocked in its catalytic site. Bacteria, left without the protection of the wall, are very susceptible to degradation by changes in the osmotic composition or pH, and die. This inhibition of proteoglycan synthesis leads to penicillins being generally more active against Gram-positive bacteria, since it is in them where the activity of DD-transpeptidase is the highest than against Gram-negative bacteria (^{Lobanovska & Pilla, 2017}). This is because Gram-negative bacteria have an additional protection called the outer membrane, which acts as a selective barrier that blocks the permeability of penicillin. In addition, Gram-negative bacteria have evolutionarily acquired the coding genes to synthesize enzymes called penicillinases or beta-lactamases, which hydrolyze (break) the beta-lactamic ring of this drug, rendering it inactive (Lobanovska & Pilla, 2017).

With the use of the first penicillins, the first bacterial counterattack occurred: the appearance of resistant Gram-positive strains. Therefore, the pharmaceutical synthesis focused on semi-synthetic penicillins that had penicellase-resistant beta-lactamic rings. An example of these are oxacillin, methylcillin and dicloxacillin, which were considered second generation penicillins. However, their spectrum of action was too narrow, therefore, in the mid-1960s, third-generation aminopenicillins or penicillins appeared, such as amoxicillin and ampicillin, which had a broader spectrum, acting on Gram-positive bacteria and Gram-negative. Even in the late 1970s, a fourth generation known as carboxypenicillins began to be synthesized. Parallel to the development of penicillin, the search for antibiotics in other organisms such as Cephalosposrium acremonium, resulted in the discovery of cephalosporins, whose combinatorial chemistry has given five generations of drugs, partly developed by the acquisition of resistance to the first generations (Table1) (^{Whitman et al., 2012}).

The origin of resistance

The causes of antimicrobial resistance (AMR) are multifactorial in nature, but the indiscriminate use of low-quality or expired drugs and lack of supervision by health authorities mainly have been recognized as the most common in enterobacteria and respiratory pathogens. In addition, the conditions of poor hygiene, the inappropriate use of antibiotics and medical malpractice, as well as the lack of regulation in pharmaceutical marketing or the lack of adherence to regulations. Almost all of these conditions are entrenched in developing countries, and it is currently here where the greatest number of microorganisms resistant to ampicillin, tetracycline and sulfonamides are found (^{Hang-Wei et al., 2016}). The circulation of strains resistant to the latter is not only due to human interaction, since the abuse in the prescription of sulfadiazine in pigs has promoted the establishment of genes involved in the degradation of sulfonamides (resistance genes) found in bacteria that make up the microbiota in the rhizosphere (part of the soil located immediately below the living roots) of maize (^{Kopmann et al., 2013}). In recent years this group of genes has been called ARG (Antibiotic Resistance Genes), of the which have been counted up to 52 ARGs present in the soil microbiota regularly exposed to fertilization with manure from cattle treated with antibiotics. Most of the bacteria present in the soil are not pathogenic. However, an issue of concern is the development and expression of AMR genes in free-living bacteria and their subsequent horizontal transfer to surrounding bacteria caused by supplementation with antibiotics in pig feed. It has been proven that soil bacteria maintain high ARGs for several months when said soil is fertilized with manure from these animals, even when a sub-therapeutic dose is handled (^{Kopmann et al., 2013}; ^{Hang-Wei et al., 2016}). Under these conditions, the arrival of a human pathogen and its Interaction with these microorganisms is catastrophic. Finally, irrigation with sewage or treated (poorly) water ends up providing the ideal microenvironment to generate super pathogenic bacteria.

Talks between bacteria: transfer of genetic information. Bacterial gatherings

Currently known as mobilome is the set of genes located in bacterial mobile elements (MGE), such as plasmids, IS elements, transposons, islands of pathogenicity and integron-associated gene cassettes (IAGC) (^{Da Silva & Dominguez, 2016}). These genes are often referred to as flexible in composition and can encode virulence factors, secretory toxins such as cholera, and antibiotic resistance. Horizontal transfer of MGE between bacteria is well documented, and therefore MGE genes may be are subject to continuous evolution and environmental changes, which can be induced or significantly accelerated by human activities.

Until a few years ago, the genus Psycrobacter was classified as an opportunistic pathogen in humans, however, this bacterium was recently identified as the cause of severe meningitis that had a fatal outcome. The antimicrobial profile found in this bacterium was quite broad (^{Ortíz-Alcántara et al., 2016}), evidencing the presence of a superbug. Another worrying example is the case of Acinetobacter baumannii (^{Kopmann et al., 2013}) with resistance to carbapenems, since this drug is perhaps the most powerful that currently exists, making it also one of the last existing therapeutic options, so the appearance of pathogenic strains with the ability to inactivate it severely limits the ability to combat and eradicate these organisms. However, it is even more worrying to find that extended-spectrum beta-lactamase enzymes (or ESBL) confer resistance to all penicillins, and also to third and fourth generation cephalosporins, as well as carbapenems, generating multidrug-resistant bacteria or superbugs, which once established within nosocomial environments, as the Acinetobacter or Pseudomonas genus inhabit, produce almost uncontrollable outbreaks (^{Morfin-Otero & Rodríguez-Noriega, 1999}).

On the other hand, the number of circulating strains of Mycobacterium tuberculosis with resistance to multiple antibiotics is increasing (^{Mbelele et al., 2018}). Derived from these interactions between bacteria and human activities, a scenario not previously foreseen with the exception of Fleming himself is developing: the combination of plasticity in the genetic exchange between free-living microorganisms (which have multiple mechanisms in horizontal transfer of information), with the antimicrobial resistance mechanisms derived from the selective pressure as the concentration of antibiotics and derivatives within the environment increases. This scenario has forced a change in priorities in health policies around the world (^{Mbelele et al., 2018}; ^{WHO, 1997}).

The new tool to identify pathogens

Research on resistance to carbapenems by Acinetobacter, Pseudomona aeruginosa and Enterobacteriaceae, as well as the incorporation of new antibiotics against them, is considered the most critical level in the WHO (World Health Organization) priority list. This is followed by the resistance reported for Staphylococcus aureus, Campylobacter, Salmonella, Neisseria and Enterococcus (^{WHO, 2020}).

The ability of whole genome sequencing of an organism, (WGS) is a high technology tool that allows the identification of these pathogens. In the 1970s and 1980s, considering obtaining the complete genome of any organism was not only overwhelming, but practically impossible and unaffordable. Back then, the technology allowed to advance to 160 base pairs per week. The sequence of a 1600 bp gene could be completed in no less than 10 weeks. But a 5 million base pair bacterial genome was impractically time consuming and expensive. The improvement brought by automated sequencers based on the Sanger method, which led to the start of the human genome sequencing project in the 1990s, incorporating 67 laboratories from around the world. It took about 13 years to sequence the 3.4 billion base pairs that our genome has, roughly at the cost of one dollar a base (^{Hernández et al., 2020}).

Currently, massive sequencers can obtain the genome of 96 bacteria in one working day. However, leaving behind the laboratory and the refinement of the molecular methods that were developed for this type of technology, it is in the development of software to analyze the genome, where the most important advances in this fight against pathogens are recorded day by day (^{Hernández et al., 2020}).

While it is true that computer technology has been a breaking point in the life of humanity for several decades, it has been in the last 20 years when a significant improvement in hardware has been achieved to think about the organization of massive data, at the same time the improvement of software for the genomic analyses has grown significantly. Current programs allow the distribution, analysis and storage of data from the complete genomes of bacteria, viruses, plants and even the human genome. The abundance of data is what marks this era, and what it demands is the efficient organization of our tools to extract information. It is in this niche where bioinformatics was born, which involves biological, chemical, statistical and mathematical elements focused on the analysis of DNA and/or protein sequences. The comparison of sequences, the identification of patterns, the ordering of variations and mutations in regions and domains of these sequences, are capable of predicting a phenotype based on statistical estimates; that is, the sequence of the complete bacterial genome can tell us how the bacterium is going to behave during an infection, and therefore how to attack it.

The first achievement was the consolidation of international databases, such as that of the ^{National Center for Biotechnology Information or NCBI} (https://www.ncbi.nlm.nih.gov/ ), which has been concentrating information for several decades and its main contribution is to have public data collections where we can find scientific articles of all kinds, genome sequences, genomic profiles, genes, point mutations, and genomic references. Indeed, we can find genes identified as responsible for antimicrobial resistance. It offers the reference materials, but the analysis is at the user's own expense (^{WHO, 2020}).

The new platforms allow users with minimal training to perform complex analysis of DNA or protein sequences, with the option for the analyzed data to be viewed by other researchers around the world and the circulation route of a microorganism can be established. For example, the ^{Comprehensive Antibiotic Resistance Database o CARD} (https://card.mcmaster.ca ), site is a database specialized in housing resistance genes, their products and associated phenotypes, it contains 3146 reference sequences, 1782 single nucleotide polymorphisms (SNPs), 2756 publications. On this basis, predictions of "packages" of resistance genes (or resistome) of about 221 pathogens have been made, and about 10272 chromosomes, 1872 genomic islands, 22692 plasmids and 213809 alleles have been identified, data that are available for comparison with the bacteria of interest and with the option to be as strict (or lax) in the comparison as the user decides (^{WHO, 2020})

At the next level of analysis is the ^{pathogenwatch}( https://pathogen.watch ), platform sponsored by the Sanger Institute in England. The site can analyze several microorganisms simultaneously, obtaining in a few minutes the identification, typing, prediction of resistance factors, and virulence. This is achieved because the platform is interconnected with several specialized databases and in a short time it can consolidate the genomic profile of a bacterium and even establish the quality with which it was sequenced (^{WHO, 2020})

The last level of analysis is not yet available for bacteria, but in viruses we can find the genome detective platform (^{Vilsker et al., 2019}). The difference with the previous ones is that it directly accepts the files with the sequencer data (fastq** format), simplifying half of the analysis. In a few minutes it analyzes the sequence of the virus, compares, typifies and characterizes. At the moment they are only available for a few viruses, but it allows a rapid, automatic and homogeneous analysis of arboviruses, influenza and SARS-COV2, speeding up the information available in cases of outbreaks and pandemics. technological developments. These types of platforms will likely be ready for bacteria and eukaryotes in a short time.

Conclusions

In the last twenty years, the World Health Organization has issued numerous alerts for the appearance of bacteria with resistance and multi-resistance to antimicrobials, especially in pathogenic organisms considered of importance in public health. If international conditions do not change significantly, it is estimated that the number of people dying in 2050 from infections of these organisms will be greater than the sum of deaths from cancer, which in 2020 amounted to 10 million people.

Worldwide, antibiotics, antiparasitics and some antivirals are losing their effect. Investment in the development of new antimicrobials is insufficient, in the same way that diagnostic tests are found to detect and isolate resistant organisms, against which, in addition, there is no vaccine in most cases.

It is expected that the strengthening in the identification and diagnosis of resistance by WGS, as well as the bioinformatics advances, can, together with the appropriate medical practice, health surveillance and the regulation of pharmaceutical marketing policies, stop and reverse the global trend that is taking place. has shown at the moment, at least in the medium term.

In the long term, advances in combinatorial chemistry, research on antimicrobial peptides of marine origin, and the use of natural bacteriophages are expected to offer new perspectives in the control of diseases caused by superbugs.