Descrição do resistoma bacteriano associado ao solo de cerrado stricto sensu

O uso e prescricao indiscriminados de antibioticos na saude humana, assim como sua utilizacao na agricultura e pecuaria, promovem a selecao de micro-organismos resistentes no meio ambiente e permite a mobilizacao e permanencia de genes de resistencia antimicrobiana no convivio humano. Alem disso, sabe-se que os antibioticos e genes de resistencia antimicrobiana tem participacao importante na sinalizacao entre micro-organismos e comunidades microbianas nos ambientes naturais. Assim, o desequilibrio na concentracao e na selecao destes genes no ambiente pode criar alteracoes importantes no fluxo de informacao entre micro-organismos com consequencias imprevisiveis na ecologia de um dado ecossistema. A colecao de genes de resistencia antimicrobiana dentro de um ambiente e denominada resistoma e um dos principais reservatorios destes genes e o solo, ambiente biodiverso e pouco abordado neste contexto. A consolidacao da metagenomica tornou possivel acessar maior quantidade de material genetico dos micro-organismos das amostras ambientais e, assim, identificar novos genes e funcoes relacionadas aos micro-organismos cultivaveis e daqueles ainda nao cultivaveis. A descricao do resistoma e a elucidacao dos mecanismos de resistencia antimicrobiana presentes no solo podem fornecer mais informacoes sobre como os micro-organismos deste neste bioma se relacionam e sobre qual o papel dos genes de resistencia . e dos antibioticos . na natureza. Duas bibliotecas metagenomicas previamente construidas com amostras de solo de Cerrado stricto sensu foram utilizadas para isolamento de clones resistentes a nove antibioticos À-lactamicos: amoxicilina, ampicilina, carbenicilina, cefalexina, cefamandol, cefoxitina, ceftazidima, penicilina G e piperacilina. Isolou-se, no total, 62 clones resistentes a cinco dos nove antibioticos testados, divididos entre as bibliotecas de pequenos e grandes insertos . nos vetores pCF430 e pCCFOS, respectivamente. Destes, selecionou-se quatro clones da biblioteca de pequenos insertos (AMX3, CFX12, CRB2 e PG17) para testes subsequentes. Os quatro clones foram testados para multirresistencia aos nove antibioticos À-lactamicos e, posteriormente, foi determinada a concentracao inibitoria minima de todos os antibioticos aos quais cada clone selecionado demonstrou resistencia. Os insertos dos clones selecionados foram sequenciados e as ORFs analisadas nos programas ORF Finder e Blastp. O sequenciamento dos insertos mostrou ORFs com funcoes relacionadas a resistencia antimicrobiana, a processos de biorremediacao de solos . com papel na degradacao de compostos aromaticos . e na sinalizacao microbiana, alem de ORFs sem funcao documentada dada por proteinas hipoteticas. Em dois insertos, identificou-se ORFs similares a enzimas transposases, o que corrobora com a nocao de que genes de resistencia estao realmente alocados em elementos moveis. A identificacao de ORFs diferentes do gpadraoh geralmente encontrado em isolados clinicos corrobora com a ideia de que genes de resistencia derivados do meio ambiente realmente possuem funcoes diversas e que, a resistencia propriamente dita, e efeito secundario destas proteinas. Tais descobertas sao promissoras ja que podem ser utilizadas no estudo do papel de alguns genes na sinalizacao microbiana e na aplicacao biotecnologica na biorremediacao de solos e amostras contaminadas. Alem disso, pode subsidiar estudos futuros sobre o real papel de antibioticos e genes de resistencia antimicrobiana na natureza e como sao transferidos do meio ambiente para o convivio humano.The misuse and over prescription of antibiotics in human health, as well as its utilization in agriculture and livestock, promotes the selection of resistant microorganisms in the environment and allows mobilization and persistency of antibiotic resistance genes in human habitat. Moreover, it is established that antibiotics and antibiotic resistance genes play an important ecological role in the environment, as they are involved in signaling processes between microorganisms and microbial communities. Hence, the unbalance in the concentration and selection of antibiotic resistance genes in the environment may alter the signaling flow between microorganisms with unpredictable implications in the ecology of a given ecosystem. The collection of antibiotic resistance genes of an environment is denominated its resistome and the main reservoir of these genes is the soil, a biodiverse and unappreciated environment in this context. The consolidation of metagenomics permitted access to a larger quantity of genetic material from environmental samples and, as such, allowed for the identification and description of new genes and functions of cultivable and not yet cultivable microorganisms. The description of the soil resistome and the elucidation of the antibiotic resistance mechanisms in this environment provide more data about the role of resistance genes and of antibiotics in nature. Two metagenomic libraries, used in the screening protocols, were previously constructed with soil samples from Cerrado stricto sensu. They were screened with the following â-lactamic antibiotics: amoxicillin, ampicillin, carbenicillin, cefalexin, cefamandole, cefoxitin, ceftazidime, penicillin G and piperacillin. In total, 62 resistant clones were isolated to five of the nine screened antibiotics, in the small and large-insert libraries. Of these, four clones from the small-insert library (AMX3, CFX12, CRB2 and PG17) were selected for further tests. The selected clones were tested for multiresistance to the nine â-lactamic antibiotics and then, the minimum inhibitory concentration was determined in all antibiotics to which each clone was resistant. The metagenomic inserts were sequenced and analyzed by the ORF Finder and Blastp programs. The sequencing reactions revealed ORFs with functions related to antimicrobial resistance, bioremediation processes roles in the degradation of aromatic and halogenated compounds and microbial signaling. They also identified hypothetic ORFs with unknown functions. In addition, two inserts exhibited ORFs similar to transposase enzymes, which correlate with the notion that resistance genes are indeed allocated in mobile elements. The identification of ORFs that are different from the pattern usually found in pathogenic isolates is compatible with the idea that environmental resistance genes possess diverse functions and that resistance per se is a secondary effect of these proteins. Such discoveries are promising as they can be raw data for assaying the role of some of these ORFs in microbial signaling and for their biotechnological applications in soil bioremediation. Futhermore, it can contribute to future studies regarding the actual role of antibiotics and antibiotic resistance genes in nature and how they are transferred from the environment to human habitat