Antibiotic Resistance: Origins, evolution and healthcare-associated infections / Resistencia a antibióticos: Origen, evolución e infecciones asociadas a la atención en salud

Abstract The increased incidences of Healthcare-associated Infections (HAI) caused by multidrug-resistant bacteria, have led to an enlarged number of morbidity and mortality cases. Besides, other factors that are affected are patients, families and institutions providing health services. Therefore, the permanent study of the subject is necessary to identify possible strategies that contribute to the reduction of the issue. A critical review of the literature based on the origin of antibiotics, the evolution of their respective resistance, and the impact on public health from a historical and current perspective was developed. The search of the literature was carried out in the bibliographic databases: Pubmed, Web of Science, Scopus, SciELO, The Cochrane Library and Lilacs. The reviewed literature showed, from the historical viewpoint, the discovery of antibiotics to the last-generation antibiotics. The rapid coevolution of genes for antibiotics resistance and its subsequent spread to hundreds of species of microorganisms by Horizontal Transfer gene (HTG) was also reviewed. It is also discussed how the expansion in antimicrobial resistance (AMR) generates a series of factors that increase health-care associated infections care (HAI) and their impact on public health. The development of antibiotics from the discovery to recent changes in the behavior and response of the microorganisms with the generation of AMR shortly after, is one of the most fantastic examples of the evolution that exists in nature.

Resumen El aumento en la incidencia de infecciones asociadas a la atención en salud causada por microorganismos multiresistentes a antibióticos, han incrementado la morbilidad, mortalidad y otros factores que afectan a paciente, familias e instituciones prestadoras de servicios de salud; por lo que se ha hecho necesario el estudio permanente del tema, para identificar posibles estrategias que contribuyan a disminuir la situación. Se realizó una revisión de la literatura sobre el origen de los antibióticos, la evolución de su respectiva resistencia, el impacto en la salud pública; desde una perspectiva histórica y actual. La búsqueda de la literatura se realizó en las bases de datos bibliográficas: Pubmed, Web of Science, Scopus, SciELO, The Cochrane Library y Lilacs. El análisis de la literatura mostró desde el punto de vista histórico, el descubrimiento de los antibióticos hasta los últimos antibióticos de última generación, y la rápida coevolución de los genes de resistencia a los antibióticos y su posterior diseminación a cientos de especies de microorganismos mediante la Transferencia Horizontal de Genes (THG). También es discutido como el incremento de la resistencia a los antibióticos (RAM) genera una serie de factores que potencian las infecciones asocia de las a los cuidados de la salud (IACS) y su impacto en la salud pública. La historia desde el descubrimiento, los cambios en el comportamiento de uso de los antibióticos y la respuesta de los microorganismos con la generación de la RAM poco tiempo después, es uno de los ejemplos más fantásticos de coevolución que existe en la naturaleza.

Resistencia microbiana desde una perspectiva metagenómica / Microbial resistance from one metagenomic perspective

Resumen Objetivo. La finalidad de esta revisión es abarcar la temática relacionada con los genes de resistencia a antibióticos, sus orígenes, reservorios y movimientos en los diferentes hábitats mediante la metagenómica funcional que permite aislar, identificar y analizar estos genes, así como el impacto que tienen en salud pública. Durante los últimos años se ha visto un gran avance en la microbiología, una de las grandes limitaciones a las que se venían enfrentado los microbiólogos era no poder acceder a la totalidad de los microorganismos que habitan el planeta. Gracias al desarrollo de diferentes disciplinas como la metagenómica se ha logrado tener el acceso a estos microorganismos. Metodología. La importancia de la metagenómica en la resistencia microbiana radica en que, actualmente, solo el 1 % de los microorganismos que habitan el suelo pueden ser estudiados por técnicas convencionales de microbiología, quedando alrededor del 99 % de estos sin estudiar. Al mitigar este gran inconveniente, la metagenómica permite el estudio de la microbiota del suelo en su totalidad generando nuevo conocimiento e información relevante en diferentes campos científicos. Resultados. Mediante la metagenómica funcional se ha podido determinar que el suelo puede ser un posible reservorio de determinantes de resistencia microbiana, debido a que la microbiota que allí habita contiene en su material genético genes de resistencia a antibióticos que confieren resistencia a un amplio espectro de antibióticos utilizados en terapia humana de forma indiscriminada y además tienen todos los mecanismos de resistencia conocidos, algunos de estos genes son generados por presión selectiva ante diferentes agentes presentes en su medio y otros son genes constitutivos que cumplen con funciones significativas en su hábitat. El gran impacto que tienen estos hallazgos está dado en que pueden representar un posible riesgo en salud pública si se adquieren por los patógenos humanos.

Abstract Objective. The purpose of this review is to cover the issues related to antibiotic resistance genes, their origins, reservoirs and movements in different habitats through functional metagenomics that allows to isolate, identify and analyze these genes, as well as the impact they have on health public. During the last years a great advance in the microbiology has been seen, one of the great limitations to which the microbiologists had been facing was not being able to have access to the totality of the microorganisms that inhabit the planet. Thanks to the development of different disciplines such as metagenomics, access to these microorganisms has been achieved. Method. The importance of metagenomics in microbial resistance lies in the fact that currently only 1 % of the microorganisms that inhabit the soil can be studied by conventional microbiology techniques, leaving about 99 % of these without studying, the metagenomics by mitigating this great disadvantage allows the study of the soil microbiota in its entirety generating new knowledge and relevant information in different scientific fields. Results. Through functional metagenomics it has been possible to determine that the soil can be a possible reservoir of determinants of microbial resistance, because the microbiota that live there contain in their genetic material antibiotic resistance genes that confer resistance to a broad spectrum of antibiotics used in human therapy indiscriminately and also have all known mechanisms of resistance, some of these genes are generated by selective pressure against different agents present in their environment and others are constitutive genes that fulfill significant functions in their habitat. The great impact of these findings is that they can represent a possible public health risk if they were acquired by human pathogens.

Perspectiva histórica del origen evolutivo de la resistencia a antibióticos / Evolutionary origin of antibiotic resistance, a historical perspective

Resumen La resistencia a antimicrobianos representa un aspecto natural de evolución bacteriana, que puede resultar de mutaciones o por adquisición de genes foráneos. Hay diferentes posturas sobre el origen de ésta resistencia que explican la habilidad de estos microorganismos de adquirir nuevas características. Las teorías de la evolución de Lamarck y Darwin, han dado pie a experimentos diseñados para explorar el origen de la variación bacteriana y surgimiento de nuevas características. Estos estudios muestran que la resistencia está relacionada con mutaciones en genes cromosomales y/o la transferencia de elementos genéticos extracromosomales, que se expresan según la presión antibiótica ejercida. Está revisión recopila los principales experimentos y las conclusiones derivadas para explicar el fenómeno de resistencia a antibióticos.

Abstract Antimicrobial resistance is a natural aspect of bacterial evolution that can result from mutations or acquisition of foreign genes. Various views on the origin of this resistance explain the ability of these organisms to acquire new features. Lamarck and Darwin's theories of evolution have led to experiments designed to explore the origin of bacterial variation and the emergence of new features. These experiments show that antimicrobial resistance is related to mutations in chromosomal genes and/or transfer of extrachromosomal genetic elements that can be expressed based on the antibiotic pressure exerted. The main experiments and findings that seek to explain the phenomenon of antibiotic resistance are reviewed here in.

Caracterization of the SOS response in Leptospira interrogans sorovar Copenhageni / Caracterização da resposta SOS em Leptospira interrogans sorovar Copenhageni

Leptospira is a basal genus in an ancient group of bacteria, the spirochetes. The pathogenic species are responsible for leptospirosis, a disease with worldwide distribution and of public health importance in developed tropical countries. L. interrogans serovar Copenhageni is the agent for the majority of human leptospirosis in Brazil. In this work, we used a great variety of experimental approaches to characterize the SOS system in this serovar, to identify its impact in general DNA damage response, as well as to assess the DNA repair toolbox owned by pathogenic and saprophytic leptospires. We identified an additional repressor LexA, acquired by lateral gene transfer, exclusively in serovar Copenhageni. We also observed that UV-C irradiation led to massive death of cells and blockage of cell division in the survivors. Both repressors were active and we identified the sequences responsible for binding to promoters. However, the LexA1 SOS box was redefined after a de novo motif search on LexA1 ChIP-seq enriched sequences. This regulator was able to bind to at least 25 loci in the genome. DNA damage also caused a massive rearrangement of metabolism: increase in expression was observed in transposon and prophage genes, in addition to DNA repair pathways and mutagenesis inducers; on the other hand, motility, general metabolism and almost all virulence genes were repressed. Two induced prophages provided several proteins with useful functions. We also assessed the DNA repair-related genes presented by the three species of Leptospira: the saprophytic L. biflexa, the facultative pathogen L. interrogans and the obligatory pathogen L. borgpetersenii. There are more diversity and redundancy of repair genes in L. interrogans in comparison with the other species. Lateral gene transfer seems to be an important supplier of DNA repair functions. In addition, leptospires share characteristics of both Gram-positives and Gram-negatives bacteria. Representative genes from several different pathways were induced during infection of susceptible mice kidneys, suggesting DNA repair genes are active while causing disease. All these data suggest mobile genetic elements are the major forces in leptospiral evolution. Moreover, during DNA damage response, several SOS-dependent and independent mechanisms are employed to decrease cell growth and virulence in favor of controlled induction of mechanisms involved in genetic variability

Leptospira é um gênero basal em um grupo já considerado um dos mais ancestrais, as espiroquetas. As espécies patogênicas são responsáveis pela leptospirose, uma doença presente em todo o mundo e de principal importância em países tropicais em desenvolvimento. L. interrogans sorovar Copenhageni é o agente da maior parte dos casos no Brasil. Nesse trabalho, utilizamos diversas abordagens experimentais para caracterizar o sistema SOS nesse sorovar, identificar seu impacto na resposta geral a danos no DNA, assim como avaliar as funções de reparo de DNA disponíveis em leptospiras patogênicas e saprofíticas. Identificamos um repressor LexA adicional, adquirido por transferência horizontal e exclusivo do sorovar Copenhageni. Observamos também que irradiação por UV-C causou significativa morte celular e bloqueio da divisão celular dos sobreviventes. Ambos os repressores são ativos e identificamos as sequências que utilizam para se ligar aos promotores dos genes regulados. Entretanto, o SOS box de LexA1 foi redefinido após uma busca de novo por motivos enriquecidos nas sequências recuperadas por ChIP-seq. Esse regulador ligou-se ao menos a 25 locais do genoma. A maioria desses alvos teve aumento de expressão após UV-C. Danos no DNA também causaram um importante rearranjo metabólico: houve aumento de expressão em transposons e profagos, além de indutores de mutagênese e vias de reparo; por outro lado, mobilidade, crescimento celular e quase todos os fatores de virulência foram reprimidos. Dois profagos induzidos durante essa resposta, possivelmente proporcionam algumas proteínas de funções importantes. Nós também avaliamos a presença de genes envolvidos no reparo de DNA em três espécies de leptospira: L. biflexa, L. interrogans e L. borgpetersenii. L. interrogans é a espécie com maior diversidade e redundância de genes de reparo. Além disso, transferência horizontal parece ser um importante fornecedor de funções de reparo nesse gênero. Leptospiras também apresentam genes característicos tanto de bactérias Gram-positivas quanto Gram-negativas. Genes representando diferentes vias de reparo foram induzidos durante infecção em modelo animal, sugerindo que essas vias estão ativas no curso da doença. Todos esses dados, em conjunto, sugerem que elementos genéticos móveis são de extrema importância na evolução do gênero e das vias de reparo. Assim, durante a resposta a danos no DNA, diversos mecanismos dependentes e independentes de SOS são empregados para frear o crescimento celular e virulência em favor da indução controlada de mecanismos para aumentar variabilidade genética

Horizontal transfer of heavy metal and antibiotic-resistance markers between indigenous bacteria, colonizing mercury contaminated tailing ponds in southern Venezuela, and human pathogens / Transferencia horizontal de marcadores de resistencia a metales pesados y antibióticos entre bacterias indígenas, que colonizan lagunas de cola contaminadas con mercurio en el sur de Venezuela, y especies patógenas para el ser humano

Bacteria colonizing heavily polluted tailing ponds in Southern Venezuela exhibit multiple resistances against mercurial compounds and antibiotics. The corresponding genetic determinants, mainly acquired through horizontal gene transfer, might also be transferred to pathogenic bacteria, an issue which represents an important risk to public health. In this work we show that indigenous, mercury-resistant bacterial strains isolated from a model tailing pond, located in El Callao (Bolivar State, Venezuela) and exhibiting a high concentration of soluble Hg, were able to transfer in vitro both heavy metal- and antibiotic resistance markers to potential human- and animal- pathogens (i.e. Escherichia coli and Pseudomonas aeruginosa). The frequencies of transfer ranged between 1.2x10-6 and 5.5x10-7 transconjugants per recipient. Transconjugants were also detected in the field, in model biofilms previously grown in natural sponges (Luffa cylindrica) and submersed in the ponds, at frequencies ranging from 1x10-4 to 5x10-3 transconjugants per recipient. These results are of particular relevance from the public health viewpoint, especially in light of the potential risk of horizontal flow of antibiotic resistance genes between indigenous bacteria and potential human pathogens.

Las bacterias que colonizan lagunas de cola altamente contaminadas en el sur de Venezuela, presentan resistencia a compuestos mercuriales y múltiples antibióticos. Los determinantes genéticos responsables de estas resistencias, adquiridos principalmente a través de transferencia horizontal de genes, pueden ser transferidos a bacterias patógenas. En este trabajo mostramos que cepas bacterianas indígenas, resistentes al mercurio y aisladas a partir de una laguna de cola modelo, localizada en El Callao (Estado Bolívar, Venezuela) conteniendo una alta concentración de Hg soluble, fueron capaces de transferir in vitro marcadores de resistencia a metales y antibióticos a cepas potencialmente patógenas para el hombre y animales (ej. Escherichia coli y Pseudomonas aeruginosa). Las frecuencias de transferencia variaron entre 1,2x10-6 y 5,5x10-7 transconjugantes por receptora. Los transconjugantes también fueron detectados en el campo, utilizando un modelo de biopelículas desarrollado en esponjas naturales (Luffa cylindrica) sumergidas en lagunas contaminadas, con frecuencias que variaron entre 1x10-4 y 5x10-3 transconjugantes por receptora. Estos resultados presentan una relevancia particular desde el punto de vista de salud pública, especialmente en vista del riesgo potencial de transferencia horizontal de genes de resistencia a antibióticos entre las bacterias indígenas y bacterias potencialmente patógenas para el hombre.

Genes y evolucinn el delgado hilo que nos conecta por miles de millones de años / Genes and Evolution, the Thin Thread that Connects Us for Billions of Years

La escala temporal en nuestra cotidianidad refleja una pequeña fracción de la historia evolutiva de los organismos, incluidos los humanos. La edad del planeta se estima en 4.500 millones de años, donde ocurrieron profundos cambios en la posición de los continentes y en el clima del planeta. Sin embargo, los cambios más drásticos surgen solo en los últimos 500 millones de años. La genética ha sido una herramienta fundamental para inferir el cambio de los organismos a lo largo de millones de años, por ejemplo, mediante la estimación de tiempos de divergencia. Los seres vivos comparten el material genético como huellas moleculares para rastrear cambios en el pasado. Así, mediante el uso de secuencias de ADN y proteínas, los científicos estudian cómo los organismos han evolucionado, cuándo surgieron importantes novedades evolutivas, cómo interaccionan los genes, y determinan las relaciones evolutivas entre las formas del pasado y las contemporáneas. Las relaciones entre organismos son inferidas por simple similaridad (fenéticas), por compartir características únicas y mediante modelos matemáticos que describen como evolucionan las secuencias de ADN y las proteínas (filogenéticas). La genética también evidencia la dinámica de los genomas (duplicación, transposición, recombinación, inversiones), que resulta en ganancia y pérdida de genes, nuevas funciones, y transferencia horizontal de genes, mecanismos que incrementan la diversidad existente hoy. Aunque el ambiente es fundamental en la evolución de los organismos, es imprescindible reconocer que hay un hilo conductor muy fino y fuerte que nos conecta inevitablemente a esas primeras moléculas que se formaron en el pasado distante.

The temporal scale of our daily life is a small fraction of the evolutionary history of organisms, including the humans. The planet age is estimated in 4.5 billion years, in which profound changes in the position of continents and the climate of the earth occurred. Nevertheless, most drastic changes arose just in the last 500 million years. The genetics has been a fundamental tool to infer the organismal change throughout billions, thousands and millions of years, for example through the estimation of divergence times. Living organisms share the genetic material as a molecular fingerprint to trace changes in the past. Thus, by using DNA and protein sequences, scientists may understand how the living organisms have evolved, when important evolutionary novelties arose, how genes interact, and determine the evolutionary relationships between past and contemporary forms. The relationships among organisms are inferred by simple similarity (phenetic relationships), by sharing unique features and throughout mathematical models that describe how DNA sequences or proteins evolve (phylogenetic relationships). Genetics also evidences the dynamics of genomes (duplication, transposition, recombination, inversions) that results in gene gain and loss, new functions, and horizontal gene transfer, mechanisms that increase today’s existing diversity. Although the environment is fundamental in the evolution of organisms, it is essential to recognize that there is a very thin and strong thread that connects us inevitably to those first molecules formed in the distant past.

Mecanismos de resistencia antimicrobiana en patógenos respiratorios / Antimicrobial resistance mechanisms in respiratory pathogens

Las infecciones del tracto respiratorio representan la indicación más frecuente de antibióticos en pacientes ambulatorios, lo cual se hace sobre bases empíricas. Una falla clínica al tratamiento puede explicarse por resistencia antimicrobiana, que últimamente ha aumentado en patógenos respiratorios. Los microorganismos desarrollan nuevos mecanismos de resistencia antibiótica y comparten virulencia por diversos sistemas. Este fenómeno representa un problema clínico y dificulta el manejo de patologías infecciosas, por lo cual es necesario utilizar nuevas estrategias para el uso adecuado de antimicrobianos. Patógenos respiratorios como S. pneumoniae y S. pyogenes presentan resistencia a betalactámicos por mutaciones en las uniones proteicas a penicilina (PBP) y la resistencia a macrólidos se explica por dos mecanismos: metilación del ribosoma y expulsión del antibiótico por bombas de eflujo;H. ínfluenzae y M. catarrhalis expresan resistencia a betalactßmicos debido a hidrólisis enzimßtica por b-lactamasas.

Respiratory tract infections are the most frequent indication of antibiotics in outpatient settings, based on empirical data. Clinical treatment failure can be explained by antimicrobial resistance, which has recently increased in respiratory pathogens. Microorganisms develop new antimicrobial resistance mechanisms and virulence is shared by different systems. This phenomenon represents a clinical problem and hampers the handling of infectious diseases. It is therefore necessary to utilize new strategies for the appropriate use of antimicrobials. Respiratory pathogens such as S. pneumoniae and S pyogenes present antimicrobial resistance to beta-lactams by mutations in penicilin binding proteins (PBP); resistance to macrolides is explained by two mechanisms: ribosome methylation and antibiotic export by efflux pumps. H. ínfluenzae and M. catarrhalis express beta-lactam resistance due to enzymatic hydrolysis by b-lactamases.

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae / Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

The extended-spectrum â-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15 percent) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital

O isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL) está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram identificadas pelo teste de sinergia em disco-duplo (DDS). Foram detectadas vinte cepas produtoras de ESBL, entre as quais Escherichia coli (n=9), Klebsiella pneumoniae (n=7), Klebsiella oxytoca (n=2) e Enterobacter aerogenes (n=2), que foram posteriormente analisadas quanto a suas características de transferência de resistência, perfil plasmidial e natureza dos genes de resistência. Os testes de transferência de plasmídios foram realizados empregando técnicas de conjugação em caldo. Os genes TEM e SHV foram analisados pela reação da polimerase em cadeia (PCR) e hibridização com sondas especificas. A detecção de plasmídios epidêmicos foi feita por análise dos plasmídios R com a enzima de restrição EcoRI. Através desta análise, foram obtidos catorze perfis plasmidiais (A, B1, B2, C1 e C2 até L).Observou-se pela PCR que a maioria dos plasmidios carregavam genes derivados de TEM e SHV, confirmados através da detecção dos genes pelos testes de hibridização. As evidencias epidemiológicas indicaram que havia uma aparente transferência horizontal dos plasmídios R conjugativos entre as enterobactérias multiresistentes neste hospital.