52a Reunião Anual da Sociedade Brasileira de Zootecnia Zootecnia: Otimizando Recursos e Potencialidades Belo Horizonte – MG, 19 a 23 de Julho de 2015 Espectro de ação de bacteriocinas produzidas por estreptococcus isolados de rúmen bovino1 Analice Claudia de Azevedo2, Isabela Maria Fernandes de Oliveira3, Bianca Lana de Sousa3, Claudia Braga Pereira Bento2, Hilário Cuquetto Mantovani4 1 Part of the máster project of the first author, funded by CNPq. Postdoc, Graduate Program in Agricultural Microbiology/UFV, Viçosa – MG. Graduate Program Student in Agricultural Microbiology/UFV, Viçosa – MG. 4 Associate Professor, Microbiology Department/UFV, Viçosa – MG. E-mail: [email protected]. 2 3 Resumo: Neste trabalho o objetivo foi avaliar o espectro de ação de bacteriocinas produzidas por 15 isolados de Streptococcus sp. obtidos de líquido de rúmen bovino. A atividade antimicrobiana foi avaliada utilizando o método de difusão em ágar e inibição direta de bactérias fibrolíticas do rúmen, bactérias patogênicas e bactérias bacteriocinogênicas. Aproximadamente 87% (13/15) dos isolados inibiram o crescimento de pelo menos um dos 17 indicadores testados. Ruminococcus flavefaciens FD1 e Lacnospira multipara D32 foram os indicadores mais sensíveis deste estudo. Streptococcus sp. C53 foi o isolado que apresentou o maior espectro de ação inibindo 7 indicadores sendo 4 produtores de bacteriocinas, duas bactérias do rúmen e uma bactéria Gram-negativa patogênica. Os indicadores, L. lactis ATCC 11454, S. mutans 1140, R. albus 7, R. albus 8, B. proteoclasticus B316, S. ruminantium GA192, L. monocytogenes ATCC 7644, B. cereus ATCC 15579 e E. coli ATCC 29214 não apresentaram sensibilidade contra nenhum dos isolados de Streptococcus sp.. Os resultados indicam que alguns dos isolados obtidos apresentaram amplo de ação, sendo capazes de inibir pelo menos um microrganismo indicador de cada grupo testado. Palavras–chave: Bactérias patogênicas, Peptídeos antimicrobianos, Ruminantes, Streptococcus sp. Action spectrum of bacteriocins produced by streptococcus isolated from rumen bovin Abstract: In this work the objective was to evaluate the spectrum of activityof bacteriocins produced by 15 isolates of Streptococcus sp. obtained from bovine rumen fluid. The antimicrobial activity was evaluated using the agar diffusion method and direct inhibition of fibrolytic rumen bacteria, pathogenic bacteria and bacteriocinogenic bacteria. Approximately 87% (13/15) isolates inhibited the growth of at least one of the 17 tested indicators. Ruminococcus flavefaciens FD1 and Lacnospira multipara D32 were the most sensitive indicators of this study. Streptococcus sp. C53 was isolated which had the largest spectrum of action inhibiting 7 indicators and four producers of bacteriocins, two rumen bacteria and Gram-negative bacteria pathogenic. The indicators, L. lactis ATCC 11454, S. mutans 1140, R. albus 7, R. albus 8, B. proteoclasticus B316, S. ruminantium GA192, L. monocytogenes ATCC 7644, B. cereus ATCC 15579 and E. coli ATCC 29214 did not show any sensitivity against isolates of Streptococcus sp .. The results obtained indicate that some of the isolates showed broad action, being able to inhibit at least one microorganism indicator of each group tested. Keywords: Antimicrobial peptide, Pathogenic bacteria, Ruminants, Streptococcus sp. Introduction The potential of rumen microrganisms for the production of enzymes and metabolites of industrial interest has increased the interest in studying these microorganisms for the production of antimicrobial substances, such as bacteriocins. These substances are proteinaceous compounds synthesized ribosomally which show varied spectrum of activity. Previous studies indicated that Ruminococcus, Butyrivibrio and Streptococcus are important producers of bacteriocins in the rumen (Russell and Mantovani, 2002). Mantovani and colleagues (2001) found that 50% of Streptococcus bovis strains obtained from bovine rumen showed antimicrobial activity against S. bovis JB1. Subsequently, the isolate showing higher inhibitory activity, named as S. bovis HC5, had its bacteriocin characterized as a lantibiotic with broad spectrum of activity. Joachimsthal et al. (2008) also found that bacteriocin production was prevalent among S. bovis isolates from ruminants in Australia and at least one bacteriocin (SB15) showed high similarity to bovicin HC5. In this work, the objective was to evaluate the spectrum of activity of Streptococcus sp. isolates previously obtained from beef cattle. Material e Methods Streptococcus sp. (n = 15) were incubated with 17 different indicators to assess the antimicrobial activity using the deferred antagonism assay. For this, the strains of Streptococcus sp. were grown in basal medium (Mantovani et al., 2001) and incubated in an anaerobic chamber for 24 hours at 39 ° C. The colonies were pretreated with chloroform _____________________________________________________________________________________________________________________________ ___________________ Página - 1 - de 3 52a Reunião Anual da Sociedade Brasileira de Zootecnia Zootecnia: Otimizando Recursos e Potencialidades Belo Horizonte – MG, 19 a 23 de Julho de 2015 and then 10 ml of basal medium (semisolid MDM or BHI media) containing the indicator culture (1% inoculum) was overlayed on top of the grown colonies. The bacteriocin was allowed to diffuse in the semi-solid agar before the plates were incubated for 24 hours in the optimum growth temperature of the indicator organism. The inhibition zones were them measured to determine the spectrum of activity of each isolate. Results and Discussion To evaluate the spectrum of activity, the bacterial isolates were tested against rumen fibrolytics, pathogenic bacteria and other lactic acid bacteria that are known bacteriocin producers (Table 1). Table 1. Deferred antagonism assay of different strains of ruminal Streptococcus sp.. The isolates were cultivated anaerobically in basal medium at 39°C and the diameter (mm) of inhibition zones was determined for to each indicator tested. Indicators Gram Isolates C4 C13 C17 C26 C28 C47 C51 C53 C57 C61 C64 C66 C70 C71 C72 HC5 Bacteriocin producers - L.lactis 11454 + - - - - - - - - - - +++ L. lactis 19453 + + - - - - - - +++ - +++ - - - - - +++ L. lactis 3147 + - - - - - - S.mutans1140 + - - - - - - - ++ - ++ - - - - - ++ - - - - - - - - - +++ S. salivarius 9 + - - - - - - - ++ - ++ - - - - - ++ S. salivarius 20P3 + ++ - - - - - - ++ - ++ - - - - - ++ R. albus 7 + - - - Ruminal bacteria - - - - - - - - - ++ R. albus 8 + - - - - - - - - - - - - - - - ++ R. flavefaciens FD1 + ++ ++ ++ + ++ - ++ ++ ++ ++ ++ ++ + ++ - +++ L. multipara D32 + ++ ++ + + ++ - ++ ++ + ++ + + ++ - - +++ S. ruminantium GA192 - - - - - - - - - - - - - - - - +++ B. proteoclasticus B316 - - - - - - - - - - - - - - - - +++ ++ Pathogenic bacteria L. monocytogens ATCC 7644 B. cereus ATCC 14579 S. aureus ATCC 25923 + - - - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - + - - - - - - - - - - - - - - - ++ E. coli ATCC 29214 E. sakazakii ATCC ++ ++ ++ 29004 - lack of inhibition zone; + inhibition zone of 1 to 7 mm; ++ inhibition zone of 8 to 14 mm; +++ inhibition zone of 15 to 22 mm About 87% (13/15) of the isolates inhibited the growth of at least one of the 17 indicators tested in this study. R. flavefaciens FD1 and L. multipara D32 were the most sensitive indicators and were not inhibited only by isolates C47 and C72. Three isolates (C4, C53 and C61) inhibited at least one bacteriocin producer used as indicador organism. E. sakazakii (gram-negative bacteria) was inhibited by isolates C4 and C53. L. lactis 11454, S. mutans, R. albus 7, R. albus 8, B. proteoclasticus, S. ruminantium, L. monocytogenes, B. cereus, S. aureus and E. coli were not sensitive to any of the ruminal Streptococcus sp.. Although some bacteriocins inhibit only phylogenetically related microorganisms (Rilley, 2011), in this work, the ruminal Streptococcus sp. showed a broad spectrum of activity. Conclusions _____________________________________________________________________________________________________________________________ ___________________ Página - 2 - de 3 52a Reunião Anual da Sociedade Brasileira de Zootecnia Zootecnia: Otimizando Recursos e Potencialidades Belo Horizonte – MG, 19 a 23 de Julho de 2015 Ruminal Streptococcus sp. showed broad spectrum of activity against different groups of pathogenic and fibrolytic bacteria. R. flavefaciens FD1 and L. multipara D32 represent useful indicators in the screening of ruminal bacteria with antimicrobial activity. Acknowledgements CNPq, CAPES, FAPEMIG References Mantovani, H. C.; Kam, D. K.; Ha, J. K.; Russell, J. B. 2001.The antibacterial activity and sensitivity of Streptococcus bovis strains isolated from the rumen of cattle. FEMS Microbiology Ecology 37:223-229. Joachimsthal, E.L.; Reeves, R. K. H.; Hung, J.; Nielsen, L. K.; Ouwerkerk, D.; Klieve, A. V.; Vickers, C. E. 2009. Production of bacteriocins by Streptococcus bovis strains from Australian ruminants. Journal of Applied Microbiology 108:428-436. Riley, M. A. 2011. Bacteriocin-Mediated Competitive Interactions of Bacterial Populations and Communities. p 1326. In Drider D, Rebuffat S (ed) Prokaryotic Antimicrobial Peptides: From Genes to Applications. Springer Science; New York. Russell J. B.; Mantovani, H. C. 2002. The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics. Journal of Molecular Microbiological and Biotechnology 4: 347-355. _____________________________________________________________________________________________________________________________ ___________________ Página - 3 - de 3
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