Bioprospecção de actinobactérias endofíticas da Mata Seca e

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UNIVERSIDADE ESTADUAL DE MONTES CLAROS 
 
Lucas Oliveira Barros 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bioprospecção de actinobactérias endofíticas da Mata Seca e Campo Rupestre 
do norte de Minas Gerais, com atividade antimicrobiana. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Montes Claros 
2019 
 
 
Lucas Oliveira Barros 
 
 
 
 
 
 
 
 
 
 
 
 
Bioprospecção de actinobactérias endofíticas da Mata Seca e Campo Rupestre do norte de 
Minas Gerais, com atividade antimicrobiana. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Montes Claros 
2019 
 
Tese apresentada ao Programa de Pós-graduação em Ciências 
em Saúde da Universidade Estadual de Montes Claros - 
Unimontes, como parte das exigências para a obtenção do 
título de Doutor em Ciências da Saúde. 
 
Área de Concentração: Mecanismos e Aspectos Clínicos das 
Doenças 
 
Orientador: Profa. Dra. Ana Cristina de Carvalho Botelho 
Coorientador: Prof. Dr. Sérgio Avelino Mota Nobre 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B277b 
 
 
Barros, Lucas Oliveira. 
 Bioprospecção de actinobactérias endofíticas da Mata Seca e Campo Rupestre 
do Norte de Minas Gerais, com atividade antimicrobiana [manuscrito] / Lucas 
Oliveira Barros. –2019. 
 61 f. : il. 
 
 Inclui Bibliografia. 
 Tese (Doutorado) - Universidade Estadual de Montes Claros - Unimontes, 
Programa de Pós-Graduação em Ciências da Saúde /PPGCS, 2019. 
 
 Orientadora: Profa. Dra. Ana Cristina de Carvalho Botelho. 
 Coorientador: Prof. Dr. Sérgio Avelino Mota Nobre. 
 
 
 1. Actinobactéria. 2. Antibacterianos. 3. Bioprospecção. 4. Bactérias Gram-
Positivas. I. Botelho, Ana Cristina de Carvalho. II. Nobre, Sérgio Avelino Mota. III. 
Universidade Estadual de Montes Claros. IV. Título. 
Catalogação Biblioteca Central Professor Antônio Jorge 
 
 
 
UNIVERSIDADE ESTADUAL DE MONTES CLAROS-UNIMONTES 
 
 
Reitor: Prof. Antônio Alvimar de Souza 
Vice-reitor: Profa. Ilva Ruas de Abreu 
Pró-reitor de Pesquisa: Prof. José Reinaldo Mendes Ruas 
Coordenadoria de Acompanhamento de Projetos: Profa. Karen Torres Correa Lafetá de 
Almeida 
Coordenadoria de Iniciação Científica: Prof. Sônia Ribeiro Arrudas 
Coordenadoria de Inovação Tecnológica: Prof. Dario Alves de Oliveira 
Pró-reitor de Pós-graduação: Prof. André Luiz Sena Guimarães 
Coordenadoria de Pós-graduação Lato-sensu: Prof. Divino Urias Mendonça 
Coordenadoria de Pós-graduação Stricto-sensu: Prof. Idenilson Meireles Barbosa 
 
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE 
Coordenador: Prof. Alfredo Maurício Batista de Paula 
Subcoordenadora: Prof.ª Marise Fagundes Silveira 
 
 
 
 
 
 
 
RESUMO 
 
 
Considerando a influência das condições ambientais reinantes nas fitofisionomias de Campo 
Rupestre e Mata Seca, bem como suas associações com o estresse hídrico e sua vinculação 
indireta com a biossintese de metabolismos secundários, definiu-se estes ambientes para este 
estudo. Adicionalmente até o presente momento não há descrito bioprospecção dos 
microrganismos nestes ecosistemas. Objetivou-se através do presente trabalho caracterizar 
populações de actinobactérias endofíticas residentes em áreas de Mata Seca e Campo Rupestre, 
com fins de obtenção de substancias antimicrobianas com potencial antibiótico. As partes 
vegetais coletadas foram esterilizadas, fragmentadas, maceradas, suspensas e cultivadas em 
meios específicos para isolamento. As colônias com características fenotípicasde 
actinobactérias foramisoladas e identificadas por Maldi-Tof MS e genotipagem do gene 16S 
rRNA. O produto metabólico das actinobactérias endofíticas do Campo Rupestre foram obtidos 
por cultivo em dois meios em caldo e separado da massa celular por centrifugação e filtração. 
O fracionamento dos produtos metabólicos foi realizado com solventes não miscíveis em água 
e o teste de atividade antimicrobiana foi avaliada por microdiluição. As amostras coletadas das 
fitofisionomias permitiram o isolamento de 16 actinobactérias endofíticas da fitofisionomia 
Mata Seca e 8 do Campo Rupestre. O Maldi-Tof MS identificou com precisão quatro isolados 
e a genotipagem do gene 16S rRNA identificou um número de 20 isolados a nível de espécie. 
Os 16 produtos metabólicos não fracionados e os 48 fracionados foram submetidos ao teste de 
inibição, sendo que, desses apenas um produto metabólico fracionado apresentou atividade 
antimicrobiana contra Staphylococcus aureus ATCC 25105 em uma baixa concentração. A 
coleção de actinobactérias endofíticas de ambas fitofisionomias apresentou uma diversidade de 
actinobactérias associada a versatilidade destas em colonizar diversas partes vegetais bem como 
as plantas em estudo. A actinobactéria Micromonospora aurantiaca, isolada do interior da raiz 
de uma planta do Campo Rupestre, produziu um produto metabólico com atividade 
antimicrobiana. 
 
Palavras-chave: Actinobacteria. Antibacterianos. Bioprospecção. Bactérias Gram-Positivas. 
 
 
 
ABSTRACT 
 
 
Considering the influence of the environmental conditions prevailing on the 
phytophysiognomies of Brazilian Rocky Field and Dry Tropical Forest, as well as their 
associations with water stress and its indirect link with the biosynthesis of secondary 
metabolisms, these environments were defined for this study. In addition, up to the present 
moment there has been no description of the microorganisms in these ecosystems. The aim of 
this work was to characterize populations of endophytic actinobacteria residing in areas of Dry 
Tropical Forest and Brazilian Rocky Field, for the purpose of obtaining antimicrobial 
substances with antibiotic potential. The collected plant parts were sterilized, fragmented, 
macerated, suspended and cultured in specific media for isolation. The colonies with phenotypic 
features of actinobacteria were isolated and identified using Maldi-Tof MS and genotyping of 
the 16S rRNA gene. The metabolic product of rockhopper endophytic actinobacteria was 
obtained by culture in two media in broth and separated from the cell mass by centrifugation 
and filtration. The fractionation of the metabolic products was performed with non-water 
miscible solvents and the antimicrobial activity test was evaluated by microdilution. The 
samples collected from the phytophysiognomies made it possible to isolate 16 endophytic 
actinobacteria of the Dry Tropical Forest phytophysiognomy and 8 of the Brazilian Rocky Field 
one. Maldi-Tof MS accurately identified four isolates and the genotyping of the 16S rRNA gene 
identified a total of 20 isolates at the species level. The 16 non-fractionated metabolic products 
and the 48 fractions were subjected to the inhibition test, of which only one fractionated 
metabolic product presented antimicrobial activity against Staphylococcus aureus in a low 
concentration. The collection of endophytic actinobacteria from both phytophysiognomies 
showed a diversity of actinobacteria associated with their versatility in colonizing various plant 
parts as well as the plants under study. The actinobacterium Micromonospora aurantiaca, 
isolated from within the root of a plant of Brazilian Rocky Field, produced a metabolic product 
with antimicrobial activity. 
 
Keywords: Actinobacteria. Anti-Bacterial Agents. Bioprospecting. Gram-Positive Bacteria. 
 
 
 
SUMÁRIO 
 
 
1 INTRODUÇÃO ........................................................................................................ 7 
2 OBJETIVOS ............................................................................................................. 15 
2.1 Objetivo Geral ........................................................................................................ 15 
2.2 Objetivos Específicos .............................................................................................
15 
3 PRODUTOS ............................................................................................................. 16 
3.1 Artigo 1: Endophytic Actinobacteria of Plants in the Bazilian Rocky Field and 
Dry Tropical Forest of Northern Minas Gerais …......................................................... 
 
17 
3.2 Artigo 2: Antibiotic Potential of Metabolic Products of Endophytic 
Actinobacteria of the Bazilian Rocky Field in Minas Gerais - Brazil ........................... 
 
31 
4 CONCLUSÕES ........................................................................................................ 40 
REFERÊNCIAS .......................................................................................................... 41 
APÊNDICES ............................................................................................................... 48 
ANEXOS ..................................................................................................................... 50 
 
 
 
7 
 
1 INTRODUÇÃO 
 
 
1.1 Microrganismos endofíticos e actinobactérias 
 
 
Os microrganismos compõem uma das maiores fontes de diversidade genética disponível entres 
os seres vivos, no entanto, esta diversidade ainda se encontra pouco descrita e explorada (1). 
Dentre os microrganismos, podem ser destacadas as bactérias, que representam um dos três 
domínios (Archaea, Bacteria e Eucarya) da árvore filogenética atualmente (2). As bactérias 
apresentam longa história evolutiva, permitindo-lhes ocupar os mais diferentes nichos da 
biosfera, sendo encontradas em praticamente todos os ambientes terrestres. As mesmas 
representam a maior biomassa viva no planeta e são responsáveis por muitos processos 
essenciais para a manutenção da vida nas condições ambientais atuais. Esta grande diversidade 
genética e metabólica faz destes microrganismos uma fonte potencial de bioprodutos (3). 
 
Além da diversidade de espécies bacterianas, existe também a diversidade de genes presente 
dentro de uma espécie (4). Observa-se que a diversidade bacteriana não é estática, sendo 
observada uma alta capacidade reprodutiva, com ciclos vitais muito curtos, o que leva a alta 
capacidade adaptativa, com rápida alteração no perfil destas comunidades devido a alterações 
do ambiente (5). Além desta grande capacidade em perpetuar a espécie, baseada na rápida 
reprodução e adaptação, a capacidade de adquirir DNA por transferência lateral de genes, 
aumenta ainda mais a diversidade e consequentemente a adaptabilidade das bactérias aos 
ambientes terrestres (6). 
 
Além das bactérias associadas às plantas na região da rizosfera, existem também as bactérias 
que colonizam o interior da planta, as quais são denominadas de bactérias endofíticas. Uma 
definição mais abrangente de bactérias endofíticas é: bactérias isoladas de tecidos de plantas 
com superficie desinfectada (7). Utilizando uma definição mais completa, temos os endófitos 
definidos como microrganismos que colonizam os tecidos internos da planta, sem causar danos 
aparentes ao hospedeiro e sem produzir estruturas externas visíveis (8). Alguns critérios para 
reconhecimento de verdadeiros endófitos foram descritos (9), onde os autores destacam a 
importância não apenas do isolamento, mas também da posterior análise de colonização das 
plantas pelos isolados candidatos a endófitos. 
8 
 
 
As bactérias endofíticas possuem, da mesma forma que patógenos, a capacidade de penetrar na 
planta e colonizar de forma sistêmica o hospedeiro, podendo habitar o aplopasto, vasos 
condutores e ocasionalmente o meio intracelular (10). Com esta colonização sistêmica da 
planta, estas bactérias podem alterar as condições fisiológicas e morfológicas do hospedeiro, 
além de atuar sobre as populações de outros microrganismos presentes no interior da planta 
(11). Muitas revisões acessam tanto o comportamento ecológico, bem como as aplicações de 
bactérias endofíticas (7, 12, 13). 
 
A atividade endofítica no controle biológico pode ser resultado de vários fatores, como 
produção de antibióticos, promoção de crescimento e indução de resistência sistêmica (14). A 
produção de antibióticos é considerada um importante fator de antagonismo nas raízes, onde 
também atuam outros compostos, como enzimas que impedem o desenvolvimento de 
patógenos. Foi constatado também o controle de patógenos por parasitismo, onde uma linhagem 
de Burkholderia cepacia, isolada de aspargos colonizou os espaços intercelulares de raízes de 
banana, levando ao controle de Fusarium oxysporum f. sp. cubense (15). Os autores observaram 
in vitro, que essa bactéria coloniza a superfície da hifa do patógeno, causando protuberâncias, 
que resultam em deformação do micélio e consequente redução no potencial patogênico do 
fungo. Em tomate, a aplicação da bactéria Pseudomonas sp. (linhagem PsJN) induziu 
resistência sistêmica a Verticilium dahliae, mostrando que a colonização do endófito é capaz 
de ativar o sistema defesa da planta, resultando na resistência ao patógeno (16, 17). Alguns 
autores sugerem que a penetração ativa da bactéria endofítica no hospedeiro, com hidrólise de 
celulose, pode causar uma reação de hipersensibilidade, ativando os mecanismos de defesa da 
planta (10). 
 
O gênero Methylobacterium também é descrito como endofítico de plantas, sendo encontrado 
em muitas espécies, como citros (18, 19), pinus (20), crotalaria (21), arroz (14) e amendoim 
(22). Além de endófitos, as espécies do gênero Methylobacterium ocupam também outros 
habitats, incluindo solo, água, superfícies de folha, nódulos, grãos, ar entre outros. Estudos 
anteriores têm demonstrado que Methylobacterium spp. coloniza ativamente a superfície de 
folhas (23). Em citros, espécies de Methylobacterium têm sido consistentemente isoladas como 
endofítico de ramos (19, 24), e nesta planta esta população interage com o patógeno Xylella 
fastidiosa (19). Espécies do gênero Methylobacterium são descritas como promotoras de 
crescimento vegetal (25) ou indutoras de resistência sistêmica (22). 
9 
 
 
A espécie M. extorquens também é amplamente encontrada em associação com as plantas, 
sendo recentemente estudada com abordagem proteômica (26), onde os autores identificaram 
proteínas exclusivamente expressas por esta espécie quando colonizando a filosfera de plantas. 
Dentre as proteínas diferencialmente quantificadas foram verificadas as presenças de proteínas 
de resposta a estresse, além de dois domínios de resposta, PhyR e um fator C-terminal, sendo 
este último referente a um mecanismo de resposta presente apenas em bactérias pertencentes à 
classe α-proteobactéria (26). 
 
As actinobactérias são bactérias filamentosas Gram-positivas, que apresentam DNA rico em 
guanina e citosina (G+C > 72%, no gênero Streptomyces e G+C de 64 a 72% no gênero 
Nocardia) possuindo a capacidade de formar hifas em algum estágio de seu desenvolvimento e 
apresentando grande diversidade de características morfológicas (27). As colônias de 
actinobactérias são formadas por uma massa de hifas, constituindo o micélio. Essa massa é 
formada a partir do desenvolvimento inicial de esporos, esporângios ou fragmentos de hifas em 
meio sólido, constituindo primeiramente o micélio vegetativo, de caráter hidrofílico. Algumas 
estirpes de actinobactérias diferenciam seu micélio vegetativo em micélio aéreo, de caráter 
hidrofóbico, o que provoca uma alteração nas características morfogenéticas, fisiológicas e 
ultraestruturas (28). 
 
Esses microrganismos estão amplamente distribuídos em ambientes naturais, como por 
exemplo, nos rios, nos mares e na atmosfera, porém o solo é o seu reservatório mais comum 
(29). Eles têm sido descritos como os principais produtores de antibióticos no solo, e também 
como um dos principais grupos microbianos produtores de enzimas de interesse comercial (30, 
31). 
 
A instabilidade genética é uma característica bastante encontrada nas actinobactérias,
podendo 
a frequência de mutação chegar a pelo menos 1 em cada 103 células no gênero Streptomyces. 
Assim sendo, a maioria das características fenotípicas relacionadas à diferenciação e 
metabolismo secundário são geneticamente mutáveis, tais como: formação de micélio aéreo, 
pigmentação, esporulação, resistência a agentes genotóxicos e resistência e/ou produção de 
antibióticos e enzimas (27). 
 
10 
 
A heterogeneidade bioquímica das actinobactérias, sua diversidade ecológica e sua capacidade 
para a produção de metabólitos secundários os fazem um bom grupo para a investiação de 
enzimas que desempenham novas atividades e/ou especificicas (32). As enzimas produzidas 
pelas actinobactérias são capazes de degradar compostos nitrogenados orgânicos, carboidratos, 
vários esteróides como colesterol, uma variedade de compostos aromáticos, acetileno e muitos 
outros. Estudos recentes vêm apontando as actinobactérias como fontes emergentes de uma 
ampla faixa de importantes enzimas de interesse industrial e ambiental (32, 33). 
 
 
1.2 Fitofisionomia - Mata Seca 
 
 
As Florestas Estacionais Deciduais (FED) são caracterizadas por um elevado grau de 
deciduidade foliar em sua estrutura arbórea e estão distribuídas pelas mais diversas regiões 
tropicais (34). Apresenta duas estações anuais bem definidas, seca e chuvosa (35, 36), que 
associadas ao potencial hídrico, temperatura e ainda suas características físicas e químicas 
permitem uma diversidade de respostas fisionômicas distintas sobre a vegetação (34). 
 
A ocorrência das FED nas regiões tropicais recebe uma denominação em escala global de: 
Florestas Tropicais Secas. No mundo inteiro, cerca de 42% das florestas tropicais se enquadram 
na definição de Florestas Tropicais Secas. Sua distribuição global pode ocorrer desde a América 
do Sul e Central até a África, Ásia e Oceania (35, 37), todavia, o conhecimento sobre elas ainda 
é limitado, implicando na necessidade de mais pesquisas (38, 39). 
 
No Brasil, as Florestas Tropicais Secas podem ser encontradas fragmentadas e isoladas ou 
imersas em zonas de transição como no nordeste, entre Cerrado e Caatinga, no norte entre 
Caatinga e Amazônia e na região centro-oeste entre o Pantanal e a Amazônia (40, 41). E 
especificamente no norte de Minas Gerais, as Florestas Tropicais Secas são habitualmente 
conhecidas como “Matas Secas”. Estas formações estão presentes dentro dos domínios do 
Cerrado e Caatinga, sendo influenciadas na sua fitofisionomia por estes biomas (42). 
 
Embora ocorram outros biomas em Minas Gerais, na região norte o bioma dominante é o 
Cerrado. Caracteristicamente o Cerrado possui diferentes fitofisionomias, como o cerradão, 
cerrado (strictu sensu), campo cerrado, campo sujo e campo limpo (43), que ainda engloba as 
11 
 
Matas Secas como parte das fitofisionomias do Cerrado. Este bioma, como todos os outros, 
vem ao longo dos anos sendo explorado exaustivamente (44-46). Uma das principais causas da 
destruição e consequente perda da biodiversidade do Cerrado é a remoção da vegetação nativa 
para a implantação de empreendimentos agrícolas e pecuários (47). 
 
As Matas Secas norte mineiras têm sofrido com essas alterações antrópicas, sobretudo sob a 
forma de atividades agropastoris. Um dos agravantes para essas áreas é que elas ocorrem em 
solos férteis favoráveis à agricultura (48), o que contribui com sua rápida ocupação. Igualmente, 
áreas de Matas Secas que em grande parte estão associadas aos afloramentos de calcário (36, 
49) também são exploradas por fábricas, principalmente as de cimentos (50). 
 
Pouca atenção era dada a esta formação fitofisionômica, entretanto, isto começa a ser 
modificado com o aparecimento de trabalhos sobre composição, estrutura e dinâmica ecológica 
das Matas Secas (38, 39, 51-53). Contribuindo assim com informações relevantes para 
compreensão dessa complexa estrutura. 
 
 
1.3 Fitofisionomia - Campo Rupestre 
 
 
Os Campos Rupestres e de altitude ocorrem, principalmente, nos topos das montanhas do leste 
do Brasil, sendo reconhecidos como importantes centros de endemismo da flora e da fauna 
neotropical (54-57). 
 
Em 1867, o botânico dinamarquês Johannes Eugenius Büllow Warming apresentou um mapa 
das regiões fitogeográficas do Brasil, no qual destacou, pela primeira vez, as vegetações de 
Campos Rupestres e de altitude como uma formação à parte do Cerrado e da Mata Atlântica, 
denominando esses tipos vegetacionais de topos de montanha mais elevados cobertos por uma 
flora alpina (58). 
 
Uma obra original de 1904 descreve a vegetação do sul do Brasil (59), apresentou informações 
sobre os campos de altitude do Itatiaia, ressaltando a ocorrência de taquaras do gênero 
Chusquea e de algumas famílias botânicas características desta região. 
 
12 
 
O naturalista mineiro Alvaro Astolpho da Silveira, um dos pioneiros nos estudos taxonômicos 
da família Eriocaulaceae nas serras brasileiras, não aplicou uma denominação específica para 
as formações abertas desta região, usando termos como “campo”, “campo limpo”, “campo 
alpestre” e “campo alpino” (60, 61). Entretanto, este autor sugeriu nomes a serem aplicados a 
certos tipos de ambientes restritos a estas regiões, tais como “chusqueal”, em referência a 
aglomerados de taquaras do gênero Chusquea nas partes mais altas da Serra do Caparaó (60), 
e “campos de eriocaulaceas”, na Serra do Cipó (61, 62). 
 
Alguns autores denominaram a vegetação aberta dos topos de montanha do leste brasileiro de 
“campos alpinos” (63, 64), possivelmente seguindo a sugestão de Gonzaga de Campos (65). 
Esses campos, na região sul do estado de Minas Gerais, já foram considerados como uma única 
unidade, sugerindo o nome de “savana especial dos altos divisores” (62, 66). 
 
O termo “Campos Rupestres” definindo a vegetação ocorrente nos topos de montanha ao longo 
da Cadeia do Espinhaço foi usado primeiramente por dois autores (67, 68). Entretanto, foi 
considerou como “Campos Rupestres” tanto o tipo de vegetação ocorrente nas partes mais 
elevadas das serras de Minas Gerais e Goiás (sobre quartzito ou arenito), quanto nos topos das 
serras do Caparaó, dos Órgãos e do Itatiaia (sobre rochas ígneas ou metamórficas), sugerindo 
que não há diferença na classificação das vegetações abertas dos topos de montanha do leste e 
do centro do Brasil (62, 68). 
 
Em geral, os Campos Rupestres ocorrem principalmente acima de 1.000 m de altitude, em 
montanhas cujas rochas são de origem pré-cambriana que foram remodeladas por movimentos 
tectônicos a partir do Paleógeno, estando associados, principalmente, a afloramentos de 
quartzito, arenito e minério de ferro (68-71). Estes campos encontram-se distribuídos 
principalmente ao longo da Cadeia do Espinhaço, embora áreas isoladas desse tipo de vegetação 
também sejam encontradas nas serras do Brasil Central (e.g., Chapada dos Veadeiros, Serras 
dos Pirineus e da Canastra) ou em montanhas da região de São João Del Rei (Serra do 
Lenheiro), Tiradentes (Serra de São José) e Itutinga, consideradas como pertencentes à Serra 
da Mantiqueira, mas com geologia e afinidades florísticas mais relacionadas aos Campos 
Rupestres da Cadeia do Espinhaço (70, 72, 73). 
 
Em geral, os Campos Rupestres da Cadeia do Espinhaço estão situados em áreas de transição 
entre o Cerrado, a Caatinga e a Mata Atlântica (71, 74). Estima-se que na porção sul da Cadeia 
13 
 
do Espinhaço, na Serra do Cipó, MG ocorra cerca de 3000 espécies vegetais identificadas, 
sendo um terço destas de ocorrência exclusiva (75). Devido às fortes pressões antrópicas 
exercidas neste ecossistema, muitas espécies de Campo Rupestre estão em vias de extinção 
(76), e já compreendem cerca de 70% das espécies de plantas consideradas ameaçadas no estado 
de Minas Gerais (77). 
 
A elevada diversidade e o alto grau de endemismos encontrados nos Campos Rupestres sempre 
estiveram associados a este mosaico de habitats
e suas singularidades, principalmente quanto 
às características dos solos que suportam esta biodiversidade (78-83). 
 
De modo geral estes habitats ocorrem em solos arenosos, fino ou cascalhentos, rasos, ácidos e 
pobres em nutrientes. A baixa fertilidade natural destes solos tem sido indicada por alguns 
estudos recentes que oferecem dados quantitativos dos teores nutricionais destes solos (78, 81, 
84-86). 
 
Além disso, estudos vêm demostrando que a baixa fertilidade natural destes solos é fundamental 
para a manutenção de espécies nativas, principalmente das consideradas endêmicas (85-87) já 
que as mesmas, em geral, estão associadas a tipos específicos do solo (86). 
 
Apesar da generalização dessa unidade florística os Campos Rupestres apresentam uma elevada 
heterogeneidade espacial incluindo um mosaico de habitats muito próximos entre si (81-83). 
Os habitats são diferenciados pela configuração do solo, continuidade da vegetação, 
composição florística, proporção de rocha exposta, presença de blocos de rocha e de sedimentos 
arenosos. Além disso, na estação chuvosa alguns destes habitats que constitui o mosaico dos 
Campos Rupestres podem permanecer secos enquanto outros permanecem encharcados, 
constituindo um sistema bastante heterogêneo (80-82). 
 
A partir da descoberta da actinomicina em 1940 e da estreptomicina, a primeira droga realmente 
efetiva para o tratamento da tuberculose, em 1943, as actinobactérias tornaram-se famosos 
como produtores de antibióticos e outros metabólitos secundários com atividade biológica. 
 
A maioria dos antibióticos empregados atualmente foi isolada de actinobactérias provenientes 
do solo. Entretanto, actinobactérias endofíticas tem-se mostrado promissores como produtores 
de antibióticos. Antibióticos de amplo espectro (munumbicinas) são produzidos por 
14 
 
Streptomyces sp. NRRL30562, um endofítico de K. nigriscans. Estes antibióticos demonstram 
atividade contra bactérias Gram-positivas, tais como Bacillus anthracis e Mycobacterium 
tuberculosis multiresistente a drogas. Munumbicina D, também é ativa contra Plasmodium 
falciparum. Streptomyces sp. NRRL30566 endofítico de Grevillea pteridifolia produz 
kakadumicina. Kakadumicina A apresenta amplo espectro, especialmente entre bactérias Gram-
positivas, também inibe P. falciparum (88). Coronamicinas, um complexo de peptídeos novos, 
foi isolado de Streptomyces sp. endofíticos de Monstera sp. e desempenha atividade bioativa 
contra Cryptococcus neoformans e P. falciparum (89). 
 
Ação sinérgica de metabólitos secundários tem sido observada em Streptomyces sp.. 
Combinações de antibióticos β-lactâmicos e inibidores da β-lactamase são conhecidos por 
serem efetivos contra bactérias resistentes a β-lactâmicos. Streptomyces clavuligerus, 
Streptomyces jumonjinensis e Streptomyces katsurahamanus todos produzem ácido 
clavulâmico e também produzem cefamicina C (um β-lactâmico). Streptomyces graminofaciens 
e Streptomyces loidensis têm sido relatados por coproduzir estreptogramina tipo A e B. 
Estreptogramina A ou B sozinha tem efeito bacteriostático e juntas têm efeito bacteriocida (90). 
 
As infecções fúngicas, além dos problemas relacionados com a resistência a compostos 
antifúngicos ainda há o relacionado com a toxidez apresentada pela maioria destes compostos. 
Na literatura a anfotericina B é aplicada no tratamento de infecções causadas por Blastomyces 
sp., Candida sp., Cryptococcus sp. e Histoplasma sp. causa nefrotoxidade, redução do fluxo de 
sangue renal, náuseas, vômito e anorexia (91). Nistatina, aplicada para candidíase também é 
tóxica no uso sistêmico e griseofulvina causa hepatotoxidade e dores abdominais. 
 
Devido à crescente aquisição de resistência por microrganismos patogênicos, a busca de novas 
substâncias com atividade antimicrobiana cresce juntamente com as pesquisas nesta área. 
15 
 
2 OBJETIVOS 
 
 
2.1 Objetivo geral 
 
 
Caracterizar populações de actinobactérias endofíticas residentes em áreas de Mata Seca e 
Campo Rupestre, com fins de obtenção de substâncias antimicrobianas com potencial 
antibiótico. 
 
 
2.2 Objetivos específicos 
 
 
 Constituir uma coleção de actinobactérias endofíticas isoladas em plantas pertencentes às 
fitofisionomiasMata seca e Campo Rupestre de Minas Gerais. 
 Genotipar as actinobactérias endofíticas obtidas. 
 Avaliar os isolados de actinobactérias para atividade antimicrobiana contra bactérias e 
levedura patogênicas a espécie humana. 
 Identificar a concentração com efeito inibitório das frações dos produtos do metabolismo. 
 
16 
 
3 PRODUTOS 
 
 
3.1 Produto 1: Endophytic Actinobacteria of Plants in the Brazilian Rocky Field and Dry 
Tropical Forest of Northern Minas Gerais. - Brazilian Journal of Microbiology enviado. 
 
 
3.2 Produto 2: Antibiotic Potential of Metabolic Products of Endophytic Actinobacteria of the 
Brazilian Rocky Field in Minas Gerais - Brazil. - Biointerface Research in Applied Chemistry 
enviado. 
 
 
17 
 
3.1 PRODUTO 1 
 
 
Endophytic Actinobacteria of Plants in the Brazilian Rocky Field and Dry Tropical 
Forest of Northern Minas Gerais 
 
Lucas Oliveira Barros1, Ronize Viviane Jorge Brito1, Ludmilla Louise Cerqueira Maia Prates1, Thaís Tiemi 
Yoshinaga1, Lorena Santos Rocha Silva1, Ana Cristina de Carvalho Botelho1, Sérgio Avelino Mota Nobre1,*. 
1 Universidade Estadual de Montes Claros, Laboratory of Epidemiology and Biocontrol of Microorganisms, Montes Claros, 
MG, Brazil 
 
* Corresponding author: S.A.M. Nobre. 
Telephone number: +55(38)999726828 
E-mail: sergio.nobre01@gmail.com 
 
 
Abstract: The Brazilian Rocky Field and Dry Tropical Forest areas in the state of Minas Gerais are important 
endemic centers, and endophytic actinobacteria were first studied in these phytophysiognomies because they make 
countless natural products, including antibiotics, antitumor agents, enzymes and immunosuppressing agents. The 
purpose of this work was to build a collection of endophytic actinobacteria that reside in Brazilian Rocky Field 
and Dry Tropical Forest areas. The collected plant parts were macerated, suspended and cultivated in specific 
isolation media. The colonies with phenotypic features of actinobacteria were isolated and identified using Maldi-
Tof MS and genotyping of the 16S rRNA gene. The samples collected from the phytophysiognomies made it 
possible to isolate 16 endophytic actinobacteria of the Dry Tropical Forest phytophysiognomy and 8 of the 
Brazilian Rocky Field one. Maldi-Tof MS accurately identified four isolates and the genotyping of the 16S rRNA 
gene identified a total of 20 isolates at the species level. The collection of endophytic actinobacteria of the Brazilian 
Rocky Field and Dry Tropical Forest phytophysiognomies presented a diversity of actinobacteria associated to 
their versatility when it comes to colonizing different plants and plant parts. 
Keywords: Bioprospection, Isolation, Actinomycetes, Cerrado 
 
 
18 
 
Introduction 
Brazilian Rocky Field (literally “rocky fields”) occur mostly on the tops of mountains in eastern Brazil and are 
acknowledged as important endemic centers of neotropical flora and fauna [1-4]. The Dry Tropical Forest of 
northern Minas Gerais are fertile regions that have been suffering with anthropic alterations, mostly due to 
agriculture and livestock farming [5], which contribute to its rapid occupation. 
Endophytic organisms are considered an important component of biodiversity. The term “endophytic” was 
coined by De Bary [6] and its definition has evolved over time; the most accurate one is “fungi, bacteria and 
protozoans that, either throughout their life or during part of it, invade the tissues of living plants and cause 
unnoticed and asymptomatic infections, but cause no disease” [7-12]. The beneficial interactions between 
endophytic bacteria and host plants have been studied before [13-15]. More recently,
studies began on endophytic 
actinobacteria as well [16,17]. 
The Actinobacteria phylum contains a wide range of Gram-positive bacteria with high guanine and cytosine 
contents (G + C) in their DNA [18]. Actinobacteria are found in natural habitats such as soil, sweet water basins, 
marine habitats, atmosphere and plant tissues [18-20], and have a remarkable ability to produce different natural 
products, including antibiotics, antitumor agents, enzymes and immunosuppressing agents [21-24]. In the past few 
years, endophytic actinobacteria have been attracting significant interest due to their ability to produce a wide 
range of secondary metabolites that can be beneficial for the host plants, promoting their growth and health 
[25,24,26]. 
Researchers are increasingly interested in the bioprospection of endophytic microbial communities that inhabit 
the plants of various ecosystems. Our purpose with this work was to build a collection of endophytic actinobacteria 
that reside in Bazilian Rocky Field and Dry Tropical Forest areas. 
 
Materials and methods 
Collecting plant samples 
The collection points for the plant parts are located in the northern region of the state of Minas Gerais, 
phytophysiognomically characterized as Brazilian Rocky Field in the municipality of Itacambira and as Dry 
Tropical Forest in the municipalities of Januária and Montes Claros, all within the state of Minas Gerais. 
Samples were collected in a random fashion, including leaf stems and whole leaves without predatory action. 
All plant parts collected were sent to the Laboratory of Epidemiology and Biocontrol of Microorganisms (LEBM) 
of the Universidade Estadual de Montes Claros (UNIMONTES) for the subsequent assays. 
 
Isolating endophytic microorganisms 
The collected plant parts were rinsed and their surfaces sterilized as described in a previously [27] then dried 
on sterile absorbent paper, after which they were fragmented and weighed. The verification of the disinfection 
process was made by inoculating three batches of 1 mL each from the last rinsing water of the samples in dishes 
containing Czapek Dox (CZP) agar and incubated at 30°C for 72 hours [28]. 
The endophytic actinobacteria were isolated by macerating the plant parts, using chemically sterilized grade 
and pistil. The maceration product was suspended in a ratio of 10-1 m/v of sterile water (ADE). Aliquots of the 
maceration product were distributed on the surface of four culture media, Nutrient Starch Ammonia Agar (MAAN) 
[29]; Starch Casein Agar (SCN) [30]; M615 Agar [31]; Czapek Dox Agar (CZP) [29] with Drigalski spatula and 
incubated at 28ºC ± 1ºC [32]. 
The colonies with characteristics typical of actinobacteria were isolated in pure cultures using successive 
subcultures in new dishes containing the original media. The incubation temperature was kept at 28ºC ± 1ºC and 
the time varied according to growth; maximum estimated time: 15 days [33]. 
The pure cultures were preserved in Czapek Dox broth with 40% glycerol and kept in an ultra-freezer at -80ºC 
[34]. 
Phenotypic characterization of actinobacteria 
The endophytic actinobacteria were cultivated and taken to micro cultivation on CZP agar as described 
previously for morphologic characterization [35]. The aerial mycelia and the reproductive structures in addition to 
the inner mycelium in the substrate were visualized using microphotography [36]. 
 
 
19 
 
Maldi-Tof MS identification of actinobacteria isolates 
The endophytic actinobacteria isolates were cultivated in CZP and incubated at 28° C ± 1º C for 15 days. A 
single fresh colony of each actinobacteria was smeared on a target steel dish with a stick. For each strain, 1 μL of 
formic acid (70%) and 1 μL of Maldi-Tof MS matrix, consisting of a saturated solution of -cyano-4-
hydroxycinnamic acid (HCCA) (Bruker Daltonics, Bremen, Germany), was applied in the area and left to dry. The 
specters were acquired using the mass spectrometer FlexControl MicroFlex LT (Bruker Daltonics) with a 60-Hz 
nitrogen laser. Before the measurements, calibration was preceded by a bacterial test pattern (E. coli DH5 alpha; 
Bruker Daltonics). The real time (RT) identification score used the criteria recommended by the manufacturer: 
scores ≥ 2,000 indicate identification at species level; scores ≥1,700 and <2,000 indicate identification at a genus 
level; scores <1,700 indicate absence of reliable identification [37]. 
Genotyping of actinobacteria isolates 
The actinobacteria were cultivated in CZP agar dishes. A cell spatula was used to extract DNA with DNeasy 
Blood & Tissue Kit (50) by Qiagen (Cat No./ID: 69504). 
The 16S rRNA genes were amplified with the primers C70 - AGAGTTTGATYMTGGC Forward and B37 - 
TACGGYTACCTTGTTACGA Reverse. Ten microliters of the raw DNA and 1 M of the primers were added to 
the reaction mixture, whose final volume was 82 L. The following conditions were used for the amplification: 
denaturation at 94º C for 45 s, annealing at 50º C for 45 s and elongation at 72º C for 45 s, with 5 s added for each 
elongation phase. A total of 25 cycles were run, followed by a final elongation phase at 72º C for 15 min. The 
pureness of the amplified product was determined by electrophoresis in 1% agarose gel (FMC Bioproducts). The 
DNA was stained with ethidium bromide and observed under shortwave UV light [38]. 
The amplified DNA was purified through precipitation with polyethylene glycol 8000. After the removal of 
Ampliwax, 0.6 volume was added (20% of polyethylene glycol 8000) (Sigma) to NaCl 2.5 M, and the mixture 
was incubated at 37° C for 10 minutes. The sample was centrifuged for 15 minutes at 15,000 x g and the sediment 
was rinsed with ethanol (80%) and sedimented as previously described. The sediment was air dried and dissolved 
in 30 mL of distilled water, then used for sequencing [38]. 
The PCR DNA sample was directly sequenced with a cycle sequencing kit (TAQuence Cycle Sequencing Kit; 
United States Biochemical Corp.). The manufacturer’s protocol was followed. The eight sequencing primers are 
shown in Table 1. The starters were marked in their extremities using 33P (Dupont, NEN) according to the 
manufacturer’s protocol. Approximately 100 ng of purified PCR DNA were used for the sequencing [38]. 
Table 1. Primers used for sequencing. 
Primers 
B12 – TGGCGCACGGGTGAGTAA FORWARD 
C31 – GGAATCGCTAGTAATCG FORWARD 
X88 – GTATTAATCACCGTTTC REVERSO 
B34 – RCTGCTGCCTCCCGT REVERSO 
B35 – GTRTTACCGCGGCTGCTG REVERSO 
B36 – GGACTACCAGGGTATCTA REVERSO 
C01 – GGTTGCGCTCGTTGCGGG REVERSO 
X91 – CCCGGGAACGTATTCACCG REVERSO 
The sequencing products generated the contigs per isolate of endophytic actinobacteria. The identity was 
assessed using the BLAST web server (http://www.ncbi.nlm.nih.gov/BLAST) [39]. The evolutive history was 
inferred through the Minimum Evolution method [40]. The percentages of the replicated trees in which it grouped 
the rates associated in the bootstrap test (1,000 copies) are displayed near the ramifications [41]. The evolution 
distances were calculated using the Kimura 2 parameter method [42] and are expressed in units of the number of 
base substitution per locus. The Molecular Evolution tree was researched using the Close-Neighbor-Interchange 
(CNI) [43] with research level 1. The Neighbor-joining algorithm [44] was used to generate the initial tree. 
Evolution analysis were carried out with MEGA X [45]. 
 
20 
 
Results 
The figures regarding the climatic conditions of the three collection points for the plant parts on the day of 
collection are represented in Table 2. 
Table 2. Sample collection points and weather parameters observed in the sample collection period. 
Collection 
pointA 
Precipitation 
(mm) 
Maximum 
temperature (ºC) 
Minimum 
temperature (ºC) 
RH 
(%) 
Location 
Januária 0.0 35.4 17.3 57.75 
S15º56'82,6'' - 
W044º45'81,9'' 
Itacambira 0.0 28.4 13.8 67.75 
S17°00'03,2''
- 
W043°33'62,6'' 
Montes 
Claros 
13.6 32.6 19.0 40.00 
S16°74'21,5'' - 
W043°89'99,9'' 
Source: INMET - Instituto Nacional de Meteorologia (www.inmet.gov.br) 
A Municipalities in the state of Minas Gerais - Brazil 
 
Plant parts of eight plants of the Brazilian Rocky Field phytophysiognomy and the roots of three of these plants 
were collected, for a total of 19 samples. Even though the collection was carried out during a season of the year in 
which it does not rain often (July), the area was very humid due to the presence of creeks. As a result, the plants 
were in a good development phase. Parts of 21 plants of the Dry Tropical Forest phytophysiognomy were collected 
between April and October; the region was in its dry season, when leaves fall or have already fallen. The result 
was 32 samples of the phytophysiognomy. The disinfection process was successful. 
After the maceration of the plant parts and the successive sub cultivations of colonies with characteristics 
typical of actinobacteria, the final yield was 263 pure cultures, of which 87 came from Dry Tropical Forest and 
176 from Brazilian Rocky Field. No antifungal agent was used in the isolation medium, as it might have inhibited 
the growth of actinobacteria; as a consequence, many filamentous fungi grew in the medium, which made it harder 
to isolate the cultures due to their quick growth in the plates. 
The microcultivation made it possible to visualize the structures that are typical of actinobacteria, which helped 
define the microorganisms with structures typical of actinobacteria. Of the 263 pure cultures of endophytic 
microorganisms, 16 were actinobacteria originally from Dry Tropical Forest and 8 of phytophysiognomies of 
Brazilian Rocky Field (Table 3). There was a higher number of isolates of endophytic bacteria of Sample2, 
belonging to the genus Lychnophora. 
More endophytic actinobacteria were isolated in the roots of the plants of the Brazilian Rocky Field 
phytophysiognomy, and in the stems of plants of the Dry Tropical Forest phytophysiognomy, due to the fact that 
more stems were collected and because of fallen leaves. 
SCN and MAAN were the best media to isolate actinobacteria, 10 and 7 isolates, respectively. The use of 
different media made it possible to acquire more isolates, but no association was observed in terms of specialization 
of endophytic actinobacteria regarding the medium used for the isolation. The M615 agar was only capable to 
isolate a member of the genus Streptomyces. 
The analysis of the collection of endophytic actinobacteria by Maldi-Tof MS was only able to accurately 
identify one isolate at the species level (Micromonospora aurantiaca) but managed to accurately identify three 
other isolates at the genus level (Nocardia, Streptomyces and Streptococcus) according to the score used that is 
significant for identification (Table 3). The other isolates of endophytic actinobacteria did not present a significant 
corresponding score for identification at the level of genus and species with the data available in Maldi-Tof MS, 
and two isolates (Act 5 and Act 20) did not show spectral peaks for analysis. 
 
 
21 
 
Table 3. Collection of endophytic actinobacteria of Brazilian Rocky Field and Dry 
Tropical Forest. 
Isolated Code Sample CodeA Phytophysiognimy Insulation mediumB 
ACT 1 Sample1 (R) Brazilian Rocky Field SCN 
ACT 2 Sample2 (H) Brazilian Rocky Field SCN 
ACT 3 Sample2 (R) Brazilian Rocky Field MAAN 
ACT 4 Sample2 (R) Brazilian Rocky Field M615 
ACT 5 Sample2 (R) Brazilian Rocky Field SCN 
ACT 6 Sample3 (F) Brazilian Rocky Field M615 
ACT 7 Sample3 (R) Brazilian Rocky Field MAAN 
ACT 8 Sample4 (H) Brazilian Rocky Field SCN 
ACT 9 Sample5 (H) Dry Tropical Forest MAAN 
ACT 10 Sample6 (H) Dry Tropical Forest MAAN 
ACT 11 Sample7 (H) Dry Tropical Forest SCN 
ACT 12 Sample8 (H) Dry Tropical Forest CZA 
ACT 13 Sample8 (H) Dry Tropical Forest SCN 
ACT 14 Sample8 (H) Dry Tropical Forest CZA 
ACT 15 Sample9 (H) Dry Tropical Forest CZA 
ACT 16 Sample9 (H) Dry Tropical Forest MAAN 
ACT 17 Sample10 (H) Dry Tropical Forest CZA 
ACT 18 Sample10 (H) Dry Tropical Forest M615 
ACT 19 Sample10 (H) Dry Tropical Forest SCN 
ACT 20 Sample11 (H) Dry Tropical Forest MAAN 
ACT 21 Sample11 (H) Dry Tropical Forest SCN 
ACT 22 Sample12 (H) Dry Tropical Forest MAAN 
ACT 23 Sample12 (H) Dry Tropical Forest SCN 
ACT 24 Sample13 (H) Dry Tropical Forest SCN 
A Relative to the plant that hosts the isolate and the sampled plant structure. (H: 
stems, F: leaves, R: roots) 
B MAAN = Nutrient Ammonia Starch Agar [29]; CZA = Czapek Dox Agar [29]; 
SCN = Starch Casein Agar [30]; M615 = M615 Agar [31]. 
 
The taxonomic identification of the isolates of the collection of endophytic actinobacteria is also in Table 4, 
which shows that 6 different phylotypes were observed in the collection. It was not possible to sequence four of 
the isolates of endophytic actinobacteria (Act 8, Act 13, Act 17 and Act 19) due to the non-amplification of the 
genetic material. However, Figure 1 shows that these isolates present morphologic structures (spores, sporangium 
or hypha fragments) typical of the actinobacteria group. 
The phylogenetic analysis showed two different groups of Streptomyces sp., one more internal and another 
more external (Figure 2). The outermost group contains strains that are more distantly related to the other groups, 
and the HBUM174061 strain of Streptomyces niveoruber was the most distant of them all. Another inner group 
formed with other species, but their sequences were not far apart. 
The phylogenetic tree in Figure 2 was improved with the sample parameters (plant of origin), plant part (H – 
stems, F – leaves, R – root) and phytophysiognomy of origin. This phylogenetic association with the parameters 
mentioned showed co-habitation of the root of Sample2 by the genus Amycolatopsis, but no evidence was found 
of the association of endophytic actinobacteria with the plant and the plant part of origin. The phylogenetic tree 
also showed the diversity of isolates of the Streptomyces genus in the collection of endophytic actinobacteria and 
highlighted this genus’s ability to colonize different plant species as well as the plants of the phytophysiognomies 
under study. 
 
22 
 
 
Figure 1. Microphotographs of the microcultivation of isolates 
Act 8 (A), Act 13 (B), Act 17 (C) and Act 19 (D), showing structures typical of 
the group of actinobacteria (the arrows indicate spores, sporangium or hypha 
fragments). 
 
The morphologic characterization by microcultivation was not accurate enough for identification, but it was 
very efficiently early on to determine whether the isolate belonged to the group of actinobacteria. Maldi-Tof MS 
proved to be a very promising technique for the identification of actinobacteria, due to its low benefi-cost ratio 
regarding DNA sequencing. The identification obtained through the two techniques shows a correspondence 
pattern of more than 50%, but the database used in the Maldi-Tof MS is still too scarce for the group of 
actinobacteria, which made it impossible to identify a higher number of species with precision. 
The taxonomic identification of endophytic actinobacteria through the sequencing of gene 16s rRNA was the 
most satisfactory method as gold standard for the identification of species: in addition to yielding a higher number 
of identifications, it also showed more reliable results. 
 
23 
 
Table 4. Taxonomic identification, through the sequencing of gene 16S rRNA and Maldi-Tof MS, of endophytic 
actinobacteria originally from the phytophysiognomies of Brazilian Rocky Field and Dry Tropical Forest of the 
northern region of the state of e Minas Gerais, Brazil. 
Isolate 
Code 
Identification through genetic 
sequencing 
Maldi-Tof identification 
Maldi-Tof 
Score A 
Act 1 Streptomyces pactum strain JG 5 Streptomyces 1.676 
Act 2 Nocardia sp. strain 7K517
Nocardia 1.795 
Act 3 Amycolatopsis sp. 102113 Amycolatopsis 1.674 
Act 4 Streptomyces sp. strain ZZ745 Streptomyces/Actinomyces/Nocardia 1.344 
Act 5 Amycolatopsis sp. R12-7 ND < 0 
Act 6 
Streptomyces olivaceus strain 
Fole3 
Streptomyces 1.531 
Act 7 
Micromonospora aurantiaca strain 
HQB393 
Micromonospora 2.137 
Act 8 ND Lactobacillus/Rhizobium 1.495 
Act 9 Streptomyces cinereoruber Streptomyces 1.569 
Act 10 
Streptomyces albus strain NRRL 
B-1811 
Streptomyces 1.53 
Act 11 Streptomyces sp. D10 Streptomyces 1.92 
Act 12 
Streptomyces niveoruber strain 
HBUM174061 
Streptomyces 1.339 
Act 13 ND Agromyces/Actinocorallia 1.347 
Act 14 
Streptomyces niveoruber strain 
173843 
Streptomyces 1.284 
Act 15 
Nocardiopsis synnemataformans 
strain BK21 
Kitasatospora/Nocardiopsis 1.369 
Act 16 
Saccharopolyspora sp. strain 
5K548 
Saccharopolyspora 1.483 
Act 17 ND Rothia 1.301 
Act 18 Streptomyces sp. ZG637 Streptomyces/Nocardia 1.597 
Act 19 ND Nocardia 1.185 
Act 20 Streptomyces sp. strain XY006 ND < 0 
Act 21 
Saccharopolyspora sp. strain 
DC11 
Streptococcus 1.913 
Act 22 
Saccharopolyspora gloriosae 
strain S79 
Streptomyces 1.293 
Act 23 Streptomyces sp. strain 16K210 Magnusiomyces 1.271 
Act 24 
Streptomyces flavoviridis strain SF 
1 
Streptomyces 1.489 
A Score value: ≥ 2.000 identification at species level; between ≥1.700 and <2.000 identification at genus level; 
<1.700 non-reliable identification. 
ND Not defined 
 
24 
 
 
Figure 2. Phylogenetic tree based on the sequences of gene 16S rRNA from the collection of endophytic 
actinobacteria resident in Brazilian Rocky Field and Dry Tropical Forest. The units of the distances are the same 
evolution units used to build the phylogenetic tree. The analysis involved 20 nucleotide sequences. The codon 
positions included were 1st + 2nd + 3rd + Non-Codifier. All ambiguous positions were removed for each pair of 
sequences. There was a total of 1598 positions in the ensemble of final data. 
 
Discussion 
Actinobacteria have a cosmopolitan distribution. They can be found anywhere, from the soil to the interior of 
plants in various ecosystems, and one study mentions the Cerrado as an important area (a hot spot) for plant 
diversity, in particular medicinal plants, sources of investigation for the isolation of associated actinomycetes [46]. 
The literature has already defined that the main invasion starting point for endophytic actinobacteria is the roots, 
from where it spreads to other parts of the host plant, [47] and it also discusses the need to explore the endophytic 
actinobacteria that live in association with plant tissues, as this is a little known area when compared with the soils 
[48-51] where these bacteria play various beneficial roles [52]. 
Some authors believe that the species, the age and the type of tissue (stems, leaves and roots) of plants, their 
geographic distribution and their location in their habitat, the season in which the sample was collected, the 
sterilization of the surface, selective cultivation media and culture conditions may all directly influence the 
isolation of endophytic actinobacteria [53-55]. 
Endophytic actinobacteria were mainly isolated from roots, followed by stems and then leaves [56,23]. Our 
study collected the root of only three plant species, but these were significant samples for the obtaining of 
endophytic actinobacteria. The fact that actinobacteria use soil as recurrent habitats supports the hypothesis of 
higher contact with plant roots, which allows them to form symbiotic associations once they enter the plant tissues 
[54]. 
We learned that using four different types of growth media was crucial for the isolation of endophytic 
actinobacteria, and the literature has described different types of media for this purpose [54,57]. The experiments 
were carried out without the use of antifungal agents in the isolation media. However, Kim et al. used 
cycloheximide and nystatin to avoid the growth of fungi during the process of actinobacteria isolation [58]. 
Actinobacteria identification is currently based on morphological, physiological and molecular studies of 
isolates [35]. Morphological identification was not satisfactory for all the endophytic actinobacteria of the 
collection. The work of Mohamed et al. made a preliminary identification of Streptomyces sp. using morphologic, 
biochemic and physiologic tests, in addition to mass spectrometry (Maldi-Tof MS) [59]. Kampapongsa and 
Kaewkla investigated the endophytic actinobacteria of rice (Oryza sativa) and analyzed the morphology of 
colonies and spores, together with the sequencing of gene 16S rRNA to identify all isolates [18]. 
25 
 
Maldi-Tof MS was not efficient in accurately identifying all the isolated endophytic actinobacteria due to the 
scarcity of information in the database regarding the group of actinobacteria, but several works use it to identify 
actinobacteria because it is a fast, low-cost and highly effective technique [60-62,59]. 
The best identification tool was the genotyping of the DNA of the isolates in the collection. Different studies 
used the sequencing of gene 16S rRNA for the phylogenetic testing and the identification of the community of 
endophytic actinobacteria of the following plant species: medicinal plant Maytenus austroyunnanensis, which 
comes from a Chinese tropical rainforest [63], medicinal plant Gynura cusimbua, which has preventive effects on 
high blood pressure, coronary disease, Alzheimer’s disease and atherosclerosis [48], Dracaena cochinchinensis 
Lour., an important plant used in traditional Chinese medicine [64], and isolates of sterilized roots of Chinese 
cabbage [65]. 
We isolated and identified 24 endophytic actinobacteria of plants of the phytophysiognomies Brazilian Rocky 
Field and Dry Tropical Forest. Qin et al. managed to isolate a total of 257 endophytic actinobacteria from roots, 
stems, leave and seeds of the oleaginous plant Jatropha curcas L.[66]. In another study of the diversity of 
endophytic actinobacteria found in the medicinal plant Maytenus austroyunnanensis from a tropical forest in 
Xishuangbanna, China, 312 isolates were obtained, which were added to the Actinomycetales order (distributed 
in 21 genera) [63]. 
Conn and Franco confirmed the presence of endophytic actinobacteria diversity in wheat roots, with the 
presence of various species of Mycobacterium and Streptomyces genera [50]. Cotin et al. investigated the 
community of endophytic actinobacteria associated to the Brazilian medicinal plant Lychnophora ericoides and 
used phylogenetic analysis to identify a predominance of the Streptomyces genus [67]. The same observation was 
made for the collection of endophytic actinobacteria in Brazilian Rocky Field and Dry Tropical Forest, after 
phylogenetic analysis. 
Studies consider endophytic actinobacteria as indirect promoters of plant growth, increased availability of 
nutrients and inducers of systemic resistance [48,68,66]. The bioactive metabolites produced by endophytic 
actinobacteria can be promising sources to fight various phytopathogens, human pathogens resistant to medication, 
and for bioremediation of the environment [63,69,68,70-72]. 
 
Conclusions 
This study was a rare investigation involving endophytic actinobacteria in the phytophysiognomies of Brazilian 
Rocky Field and Dry Tropical Forest, belonging to the Brazilian territory. It became evident that the endophytic 
microbial populations in the two phytophysiognomy are diverse and expressive and may be associated to the water 
stress to which the host plants were submitted in these habitats. Actinobacteria, in particular the Streptomyces 
genus, are versatile and can colonize different plants and different plant parts in the phytophysiognomies under 
study. The diversity of the endophytic actinobacteria of the collection is associated to a variability in the 
composition of the media
used for isolation. 
26 
 
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3.2 PRODUTO 2 
Page | 32 
 
 
 
 
Antibiotic Potential of Metabolic Products of Endophytic Actinobacteria from Brazilian 
Rocky Field in Minas Gerais - Brazil 
Lucas Oliveira Barros1, Ronize Viviane Jorge Faria1, Jotta Junior Novaes1, Ana Cristina de Carvalho 
Botelho1, Sérgio Avelino Mota Nobre1,* 
1 State University of Montes Claros 
*corresponding author e-mail address: sergio.nobre01@gmail.com 
ABSTRACT 
The emerging resistance of pathogens to the available therapies underscores the need to discover new antimicrobials and endophytic 
actinobacteria attract significant interest in their ability to produce a large number of secondary metabolites exhibiting a variety of 
biological activities. We aimed to obtain fractions of the metabolic product of resident endophytic actinobacteria from the Brazilian Rocky 
Field with antimicrobial potential. The metabolic product of rockhopper endophytic actinobacteria was obtained by culture in two media 
in broth and separated from the cell mass by centrifugation and filtration. The fractionation of the metabolic products was performed with 
non-water miscible solvents and the antimicrobial activity test was evaluated by microdilution. The 16 non-fractionated metabolic products 
and the 48 fractions were subjected to the inhibition test, of which only one fractionated metabolic product presented antimicrobial activity 
against Staphylococcus aureus in a low concentration. The actinobacterium Micromonospora aurantiaca, isolated from within the root of 
a plant, produced a metabolic product with antimicrobial activity. 
Keywords: Actinobacteria, Anti-Bacterial Agents, Gram-Positive Bacteria 
1. INTRODUCTION 
 The phylum Actinobacteria comprises a wide variety of 
Gram-positive bacteria with high content of guanine and cytosine 
(G + C) in DNA [1]. Actinobacteria are found in natural habitats, 
such as soils, freshwater basins, marine habitats, atmosphere and 
plant tissues [1-3], and are notable for their ability to produce 
various natural products, including antimicrobials, antitumor 
agents, enzymes and immunosuppressive agents [4-7]. 
 In recent years, endophytic actinobacteria have attracted a 
significant interest in their ability to produce a large number of 
secondary metabolites that exhibit a variety of biological activities, 
such as antimicrobials, antitumor agents, plant growth promoters 
and enzymes, and can contribute to plants hosts promoting growth 
and health [7-9]. 
 Deaths attributable to antimicrobial resistant infections are 
expected to rise to more than 10 million by 2050, this emerging 
resistance of microbial pathogens to the available therapies, and the 
current increase in the number of new diseases underscores the need 
to discover new antimicrobials to combat infectious diseases [10]. 
We aimed to obtain fractions of the metabolic product of resident 
endophytic actinobacteria from the Brazilian Rocky Field with 
antimicrobial potential. 
2. EXPERIMENTAL SECTION 
 
Figure 1. Sequence of the activities of the experiments. 
Collection of microorganisms 
 Endophytic actinobacteria were obtained from the 
Laboratory of Epidemiology and Biocontrol of Microorganisms 
(LEBM) of the State University of Montes Claros (UNIMONTES). 
Obtaining metabolic product 
 Endophytic actinobacteria were cultured on plates 
containing Czapek Dox agar (CZP) [11] at 28 ± 1°C for 15 days. 
After the incubation time 3 ml of sterile water was added to the 
plates to suspend the mycelium with the spores. Transfer the 
suspension to erlenmeyers containing Amido Nitrate Casein (SCN) 
[12] and Czapek Dox (CZP) broth in 1:20 ratio, remained under 
agitation for 12 hours at 150 rpm and 12 hours of rest for 15 days at 
28°C [13]. The metabolic fluid was separated from the cell mass by 
centrifugation at 10,000 rpm for 15 minutes. The complete 
separation of the two phases was done by filter membrane (0,22 
m) [14]. 
Fractional collection of metabolic product 
 The extraction of the active principle from the metabolic 
residue was carried out with non-water miscible solvents (ethyl 
acetate, chloroform and ethyl ether). Used the ratio of 2: 1 solvent / 
Volume _, Issue ..., 201_, ...-... ISSN 2069-5837 
Open Access Journal 
Received: / Revised: / Accepted: / Published on-line: 
Original Research Article 
Biointerface Research in Applied Chemistry 
www.BiointerfaceResearch.com 
Antibiotic Potential of Metabolic Products of Endophytic Actinobacteria of the Bazilian Rocky Field in Minas Gerais - Brazil 
Page | 33 
metabolic product with shaking at 150 rpm for 20 minutes. After 
the elapsed time, the two phases were separated by centrifugation 
and the solvent part was removed for drying and obtaining the dry 
active principle. The dry active principle was suspended only with 
sterile water for inhibition tests [13]. 
Antimicrobial activity of metabolic products 
 The antimicrobial activity of the non-fractionated and 
fractionated metabolic residue of the endophytic actinobacteria was 
evaluated in 96-well microdilution plates against Escherichia coli 
ATCC 8739; Pseudomonas aeruginosa ATCC 27853; 
Staphylococcus aureus ATCC 25105; Candida albicans ATCC 
10231. 
 The inoculum of the test bacteria was adjusted in 0.85% 
saline to 0.5 McFarland 0,5 (1,5 x 108 UFC/mL), diluted 1:10 to 
obtain a concentration of 107 UFC/mL. When 5 L of this 
suspension was transferred into 95 L of Mueller Hinton broth a 
final concentration of 5 x 105 UFC/mL. Each well of the 
microdilution plate was inoculated a volume of 10 μL of inoculum 
into 90 μL of metabolic residue [15]. 
 The yeast inoculum was adjusted in 0.85% saline to 0.5 
McFarland (1 x 106 cells/mL), diluted 1: 100 also in saline, followed 
by a 1:20 dilution in RPMI 1640 liquid medium, resulting in in a 
final concentration of 5 x 102 to 2.5 x 103 cells/mL, this being the 
test concentration. Each well of the microdilution plate was 
inoculated in a ratio of 1:1 (100 μL of inoculum in 100 μL of 
metabolic residue [15]. 
Determination of minimum inhibitory concentration by 
microdilution 
 The minimum inhibitory concentration (MIC) determination 
was performed by microdilution in 96-well plates, the inoculum of 
the microorganisms tested was done as previously described and the 
test followed the standard determined by the manual Clinical and 
Laboratory Standards Institute (2010) [15]. The metabolic residue 
concentrations used in the test were: 100%; 50%; 25%; 12.5%; 
6.25% and 3.125% from the origin. The microplates were cultured 
at 37 ° C for 18-24 hours. 
 After the incubation time, the 96-well plates were developed 
with 20 μL of resazurin (0.02%) and incubated for 2 hours to 
observe the occurrence at the change in staining, the blue color 
indicated inhibition of microbial growth and pink color indicated 
microbial growth. The MIC was defined as the lowest concentration 
of fractionated