Evolution of AMR

Since the emergence of humankind, the use of natural resources ha sbeen sought for its benefit, as an adaptive strategy to different environments.Several natural products were used by observation or intuition, in order to improve their health and welfare, mainly, facing the presence of pathogenic agents. Once these resources were considered exhausted and measured by advances in science, they resorted to chemical synthesis ¹⁰.

AMR has been described since the beginning of the 30s. After the use of penicillin in World War I, the first resistant bacteria emerged; in 1945, Fleming postulated the potential risks associated to the use of antibiotics; he showed that the use of a large and prolonged scale can select resistant bacteria, observing in his laboratory that bacteria sensitive to penicillin multiply in the presence of increasing concentrations of the antibiotic ¹¹. During the 40s, the first report of penicillin resistance by strains of Escherichia coli (E. coli) and Staphylococcus sp was reported ¹². In 1947, resistance to streptomycin among patients with tuberculosis was detected, where 80% of them relapsed within three months due to the formation of resistant bacilli ¹³. In the years 1952 and 1957, resistance to tetracycline and chloramphenicol was reported and in the decade of the 60s, β-lactamases producing strains, such as TEM and SHV of wide spectrum (detected in gram-negative bacilli) were discovered ^14,15.

Extended-spectrum β-lactamases (ESBLs) are phenotypically resistant to penicillins and cephalosporins; they were registered for the first time in E. coli in 1964 ¹⁶. Later, in the 80s, antibiotics such as aminoglycosides (including vancomycin) were detected from resistant strains of Enterococcus; a short time later, it was found resistance to ampicillin in different species and the list kept growing ¹⁷. At the end of the 70s, bacteria resistant to ampicillin and cephalosporins were reported ¹². In 1980, it was estimated that between 3-5% of Streptococcus pneumoniae (S. pneumoniae) was resistant to penicillin; but in 1998, 34% of these bacteria increased their resistance to this antibiotic. In the same decade, resistance to vancomycin and erythromycin was observed ¹³. Subsequently, in 1999 the multiresistance of gram-negative bacteria was described. For the period from 2002 to 2009, an increase in strains of E. coli resistant to broad-spectrum cephalosporins was observed, which is present in most of European countries. In 2008, a new enzyme called "New Delhi Metallobe-talactamase" was observed, which confers resistance to all beta-lactam antibiotics, except aztreonam; showing a global alert against AMR to several non-beta-lactam antibiotics ¹⁸, leaving a few therapeutic options for the treatment of patients infected with these bacteria. After this report, the presence of this type of resistance was identified in 2010 in Canada, in 2011 in the United States and Guatemala; in 2012 in Uruguay, Paraguay and Colombia ¹⁹.

AMR is one of the most spectacular and documented natural event in microbial evolution, from the origin and application of antibiotics, passing through a few years in order to make that the different phenotypes arise; twelve years after the origin and application of penicillin, the first resistance mechanism was detected ²⁰. This is how it is observed that, in a few years, bacteria can increase the speed of AMR generation. For example, in ten years after having resistant strains to penicillin and methicillin, resistance to chloramphenicol emerged, and in four years to streptomycin. Therefore, AMR is a growing public health problem, seen barely some years after the discovery of penicillin ²¹. That is why, a question arises from the emergence of AMR: What is or what are the mechanisms involved in this fast production of AMR?

Origin of AMR

Throughout the five decades that followed the synthesis and the indiscriminate use of antibiotics in people, animals and agriculture; a selective process unprecedented in the history of evolution has been observed, due to the fact that it has been an unregulated practice that lacks control and supervision ²²; but this selection, considered by many researchers as artificial, has many components to consider in this review; in addition to the selective effect, the high rate of mutations in bacteria, the formation of bacterial communities and the horizontal transfer of genes, are important factors in the generation of AMR ²³. There are several examples in the literature of spontaneous mutations in bacteria; some researchers consider that the evolution of resistance, through the acquisition of spontaneous mutations is particularly relevant for certain drugs, such as quinolones and rifamycins, for which the high-level resistance can result from a point mutation ^22,24. For example, in Salmonella typhimurium (S. typhimuriun), with a point mutation in the henC gene, the resistance of the bacteria to protamine increases, but with a cost in the reduction in bacterial growth ²⁵; similar results were described in Salmonella enterica, where mutations in the tRNA-isoleucine gene confer resistance to mupirocin but with a reduction in growth ²⁶. Other authors consider that AMR can evolve through the accumulation of multiple sequential mutations and not by single point mutations ²⁷; this mechanism would be responsible of the high levels of AMR that currently present many of the microorganism species, pathogenic species ²⁸. These results, have shown that microorganisms that have a strong selective pressure (high concentrations of antibiotics), have RAM in a short period; similar to the one presented currently, especially in treatments against infections in humans ²⁹.

It is presumed that there exist around 20 thousand resistant genes, predicted through the analysis of DNA sequences of different bacterial genomes however, they are functionally expressed in just some of them ²⁸; many of them are originated by unique or consecutive punctual mutations, or also by gen duplications. But, what is the reason for these genes to be distributed in other strains or bacterial species presented in different environments? The answer to this question can be found in studies of comparative genomics. The identification of the sequences of bacterial genes in eukaryotic genomes, as the presence and genomics of pathogenicity islands presented in E.coli, found in other animal pathogenic, in human genome and some plant species; they confirm the theory of horizontal gene transfer ³⁰. Horizontal gene transfer (HGT) has been considered as the mechanism responsible of the dissemination of antimicrobial resistance genes through different bacterial species ³¹. Actually, the genes that present resistance to certain antibiotics in non-related phylogenetically bacteria, demonstrate to have identical nucleotide sequences, including Gram positive and negative bacteria; it emerged at the beginning of the decade of 1990; it was a way to explain the phylogenetic incongruence using different gene trees. This process can also occur among the domains in all the possible directions, from bacteria to archaea, bringing new data about the rise of the genomic era, which has permitted the comparison of genes among different species ³². The interchange of genetic material in HGT among genomes is carried in different ways, acquiring a great relevance in the prokaryotic evolution due to the resistance to antibiotics that contribute to the inclusion of new mechanisms by bacteria ^33,34.

HGT is a phenomenon which takes place in and within the three domains of life (Fig. 1). The acquisition of genes by bacteria has got accelerated by the increase of adaptive and selective pressure needs, specifically the use of antibiotics in infections control in medicine, veterinary, agriculture and animal nutrition ³⁵; the mark of the transference corresponds to the existence of a gene or genic sequence in the phylogenetic tree of the organism and to the observation of the same genic disposition in the donor and receptor bacterial population ^31,36.

Las HSPs tienen 4 regiones funcionales conservadas. En Azul claro dominio N-terminal (llamado dominio J), En azul la región flexible rica en glicina/fenilalanina, en verde la región M de unión al sustrato y en rojo la región N-terminal. Fuente: Realizada por los autores de la revisión.

Figure 1 Evolution of antibiotics synthesis 

With the recent increase of the studies in metagenomics, in which resistance to antibiotics has been identified in different ecosystems ³⁷, for example; in human micro biome which generated complete genome sequences of several hundreds of human microbes, it has confirmed this HGT theory. Liu et al, detected a total of 13.514 genes coming from HGT identified in 308 human microbes in different parts of the body (including intestine, mouth, skin, etc.), with an average of 43,9 HGT per microbe THG ³⁰. Besides this finding, researchers discuss the possibility of THG among the micro biome and the cells of our body; and how this event can be related to human health due to the fact that the total number of microbial cells hosted by the human body is 10 times greater than the number of human cells in the body (100 times the number of genes in the human genome); The theory of HGT between the microbiome and the cells of our body is more than supported, but this behavior is not exclusive of the human microbiome ³⁸.

The acquisition of genes by bacteria is accelerated, increasing the need of adaptation and selective pressure, specifically, by the use of antibiotics to control infections in human medicine, veterinary medicine and agriculture. Therefore, being in permanent contact with diverse environments, farm or domestic animals, plants, insects, among others HGT could be present even more frequently than it is commonly thought ^31,39

Among the most probable mechanisms of HGT are conjugation, transformation and transduction; in which mobiñe genetic molecules take part such as plasmids, bacteriophages, transposons, integrons and gene cassettes that have genes with functions for their own transfer and / or bacterial resistance ⁴⁰. In chat 1 examples can be found of mobile genetic elements that transfer resistance genes.

Chart 1 Examples of mobile genic elements that transfer resistance genes 

One of the most common and known mechanism is the conjugation by means of plasmid transfer, taking resistance genes; in Gram-negative bacteria, resistance genes are found as a part of small mobile genetic elements or "cassettes", integrated in greater elements (integrons) ⁴¹. Integrons are structures of interest because they are found in the bacterial chromosome structure presented in the cassettes of genes related to resistance; it has been observed that more than a cassette can be inserted in the same integron to generate molds that contribute to the spreading of the multiple resistance ⁴². Resistance genes spreading is higher when these are part of mobile genetic cassettes, which permit them to be transferred by several mechanisms ⁴³. There exists enough scientific evidence of the high rate of HGT among gram-negative and positive bacteria, generated mainly by conjugation.

Cassettes can codify several compounds that generate resistance for a huge range of antibiotics including 6-lactam, aminoglycosides, trimethroprim, amphenicol, sulfonamide, tetracyclines, rifampicin, erytromycin and quinolones ⁴⁴.Therefore, integrons and cassettes that bring multiple ARM are, currently, the most studied genic elements by researchers in order to explain the origin of ARM and its impact in public health.

AMR in health-care associated infections

During more than 60 years, antibiotics have been considered as the panacea to cure infections, with enormous benefits for human health. The development of the resistance to this important class of medicaments, and the consequent loss of its efficacy as an antimicrobial therapy, represents a serious health menace. Despite the efforts of hospitals to improve the caring process and health of the patient, infections still occur with a higher frequency; it has been complex to determine the world range exactly; it is estimated that every year billions of patients get affected ⁴⁵. Health-care associated infections (HAI) are defined as any infectious process, general or localized, that occurs due to the stay or attendance to a health center and appears during or after the discharge more than 48 hours after the entry. They include blood infections, affected area by a surgery, skin and soft tissue, pneumonia, and urinary tract infections which are the most common ⁴⁶. Therefore, HAI, besides entailing an adverse effect for the patient, are also an indicator of the caring quality. The rise and reemergence of HAI, caused by ARM microorganisms, has as a consequence the increase of morbidity and mortality in hospitals around the world ^47,48. They are associated to economic effects in institutions, in health systems and therefore in economical ranges for the countries ^49-51.

In the American continent, a prevalence of HAI is present which varies from 4.5% in the United States, to 14% in Brazil ⁵². Other studies in several countries of America reveal a wide variation in the incidence of resistance in common bacterial pathogens, as an example, the resistance to third generation cephalosporines observed in E. coli, varying from 0% in the case of Brazil to 50% in Peru; in comparison with the world reports where they are found in 26.8% ⁵³.

The increase of the resistance has become one of the most important aspects in the world and this is why, the antimicrobial resistance was declared as a public health problem by the World Health Organization in 1999; it is related to the excessive and indiscriminate use of antibiotics in the community and hospitals, as a decisive factor in the origin of the rise of resistant pathogens nowadays ³.Therefore, it is necessary to promote strategies of control of AMR through the exact identification of the microorganism and its resistance phenotype, besides the opportune information of these results to the service of infectious disease treatment in the hospital, in order to avoid the proliferation of multiresistant strains that produce new HAI ⁴⁶.