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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Potencial bacteriocinogênico de Streptococcus sp. isolados de rúmen de bovinos1
Bianca Lana de Sousa1, Isabela Maria Fernandes de Oliveira 1, Analice Claudia de Azevedo2, Cláudia Braga Pereira
Bento2, Hilário Cuquetto Mantovani3
1
Part of the máster project of the first author, funded by CNPq.
Graduate Program Student in Agricultural Microbiology/UFV, Viçosa – MG.
Postdoc, Graduate Program in Agricultural Microbiology/UFV, Viçosa – MG.
4
Associate Professor, Microbiology Department/UFV, Viçosa – MG. E-mail: [email protected].
2
3
Resumo: Nesse trabalho o objetivo foi avaliar a produção de bacteriocinas em isolados de Streptococcus sp. do
rúmen. Streptococcus sp. foram isolados de três bovinos de corte alimentados com feno de tifton 85 (75 culturas) e
de três vacas leiteiras alimentadas com silagem de milho (75 culturas). A atividade antimicrobiana foi avaliada in
vitro pelo método de difusão em ágar e inibição direta de S. bovis JB1 e S. bovis HC5. Os genes de biossíntese de
lantibióticos lanB, lanC e lanM e os genes estruturais das bacteriocinas bovicina HC5 e bovicina 255 foram
amplificados por PCR. Em 34% dos isolados de Streptococcus sp obtidos do rúmen de bovinos de corte foi
identificada a atividade inibitória contra pelo menos um dos indicadores testados. S. bovis JB1 foi mais sensível à
atividade antagonista do que S. bovis HC5. Nas culturas de Streptococcus sp. isoladas das amostras de líquido
ruminal das vacas leiteiras não foi identificada inibição contra nenhum dos indicadores testados. Os genes de
biossíntese foram encontrados tanto em isolados que apresentaram atividade antimicrobiana quanto em isolados que
não demonstraram capacidade de inibir as culturas indicadors. Os isolados de gado de corte apresentaram maior
diversidade de genes de biossíntese em relação aos isolados obtidos de vacas leiteiras. A presença do gene
estrutural da bovicina HC5 e da bovicina 255 foi identificada em 10 e 28 dos isolados testados, respectivamente.
Palavras–chave: Bacteriocinas, bovicina HC5, bovicina 255, gado de corte e vacas leiteiras.
Bacteriocinogenic potential Streptococcus sp. isolated from bovine rumen
Abstract: In this study the objective was to evaluate the production of bacteriocins in isolates of Streptococcus sp.
from rumen. Streptococcus sp. were isolated from three beef cattle fed Tifton 85 hay (75 cultures) and three dairy
cows fed corn silage (75 cultures). The antimicrobial activity was evaluated in vitro by the agar diffusion method
and direct inhibition of S. bovis HC5 and S. bovis JB1. Lantibiotics biosynthesis genes lanB, lanC and lanM and
structural genes of bacteriocins bovicin HC5 and bovicin 255 were amplified by PCR. In 34% of the isolates of
Streptococcus sp obtained from the beef cattle rumen has been identified inhibitory activity against at least one of
the tested indicators. S. bovis JB1 was more sensitive to antagonist activity than S. bovis HC5. In cultures of
Streptococcus sp. isolated from rumen fluid from dairy cows was not identified inhibition against any of the tested
indicators. The biosynthetic genes were found in both isolates showed antimicrobial activity and in isolated not
demonstrated ability to inhibit the indicadors cultures. Beef cattle isolates showed greater diversity of biosynthetic
genes in relation to isolate obtained from dairy cows. The presence of the structural gene bovicin HC5 and bovicin
255 was identified on 10 and 28 out of the isolates tested, respectively.
Keywords: Bacteriocins, bovicin HC5, bovicin 255, beef cattle and dairy cows.
Introduction
The genus Streptococcus presents many bacteriocin-producing species and some studies to identify new
antimicrobial peptides have focused on this group of bacteria to screen for biosynthetic genes, such as lanB e lanC
(Marsh et al., 2010). Among the ruminal bacteriocin-producing species Streptococcus equinus (Streptococcus
bovis) has been described as a potentially useful organism for manipulation of rumen fermentation as an alternative
to classical antibiotics (Russell and Mantovani, 2002). In this regard, bovicin HC5, a lantibiotic with large spectrum
of activity produced by S. equinus HC5 has been shown inhibitory activity against hyperammonia-producing
bacteria and methane formation in vitro (Lima et al., 2009, Lee et al., 2002). However, studies to identify other
potentially useful bacteriocin-producing streptococci from the rumen ecosystem have not been carried out. In this
study the aim was to isolate ruminal Streptococcus sp. from beef and dairy cattle and to determine their
antimicrobial activity in vitro. Additionally, the presence of genes involved in the biosynthesis of antimicrobial
peptides (lantibiotics) were investigated in these bacterial isolated using PCR.
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Material e Methods
The cultures of Streptococcus sp. were isolated from rumen fluid samples collected from three fistulated
dairy cows fed corn silage ad libitum containing 7.29% crude protein (dry matter basis) and three fistulated beef
cattle fed Tifton 85 hay (Cynodon sp. ) ad libitum containing approximately 4.6% of crude protein (dry matter
basis). All isolates (n = 150) were initially screened by deferred antagonism assays to evaluate their antimicrobial
activity against the indicator bacteria S. equinus HC5 (bacteriocinogenic culture) and S. equinus JB1 (nonbacteriocinogenic culture). The biosynthetic genes involved in the modification of lantibiotics (lanB, lanC and
lanM) and the structural genes encoding bovicin HC5 (BvcAa) and bovicin 255 were amplified using PCR and the
genomic DNA extracted from the isolates. Genetic diversity of the isolates was characterized using BOX-PCR and
the polymorphism of bands was compared in Bionumerics 5.1 software using Dice similarity coefficient.
Results and Discussion
Isolates were grown anaerobically on basal medium plates and an overlay containing S. equinus HC5 or
JB1 (approximately 107 CFU/ml) was performed on top of the grown colonies after 24 h of incubation. Appearance
of zones of clearing was used as an indication of antimicrobial activity. Approximately 34% of the isolates obtained
from beef cattle (26/75) inhibited S. equinus HC5, S. equinus JB1 or both (Figure 1).
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Figure 1. Genetic diversity and antimicrobial activity of Streptococcus sp. Isolated from beef cattle (A)
and dairy cows (B). The cladogram represents the BOX_PCR pattern of the isolates and the boxes represent the
presence of biosynthetic genes involved in bacteriocin production. Symbols +/– indicate positive or negative results
for antimicrobial activity against S. equinus HC5 and S. equinus JB1.
Fifteen isolates presented activity against both indicators and the size of the inhibition zones was greater
(P<0.01) against S. equinus JB1 (non-bacteriocinogenic) compared to S. equinus HC5 (bacteriocinogenic). None of
the isolates obtained from dairy cows was able to inhibit any of indicators tested in this study (Figure 1). Because S.
equinus JB1 was always more sensitive than S. equinus HC5, it appears that immunity mechanisms found in
bacteriocinogenic cultures could confer cross-resistance to similar peptides and greater ecological advantage against
competitors.
A variety of PCR products were obtained when BOX-PCR was performed using DNA extracted from
ruminal Streptococcus sp.. The PCR products varied in size from approximately 300 to 5000 bp. Twenty five
isolates from beef cattle and twenty cultures obtained from dairy cows presented 100% similarity of (Figure 1)
being considered as isolated clones.
At least one biosynthetic gene was identified in 82% of the bacterial isolates obtained from beef cattle,
while 60% of the cultures isolated from dairy cows showed at least one biosynthetic gene. Fourteen and twenty six
isolates from beef cattle and dairy cows, respectively, contained only lanM, suggesting that this isolates are
potential producers of class II lantibiotics, which are modified posttranslational by the bifunctional enzyme LanM.
The genes lanC and lanM were identified together in only ten isolates from beef cattle. Thirteen isolates from beef
cattle presented the genes lanB, lanC and lanM concomitantly in their genomes. The amplification of genomic
DNA using primers targeting the structural gene encoding bovicin HC5 and bovicin 255 resulted in the
identification of 10 isolates containing the gene bvcAa and 28 isolates with the bovicin 255 gene. The identification
of the structural gene encoding bovicin HC5 and bovicin 255 in several ruminal bacteria suggest that variants of
these peptides might exist among ruminal streptococci.
Conclusions
Bacterial isolates showing distinct antimicrobial activities were obtained from beef cattle and dairy cows
suggesting ecological differences imposed by diet or the host on the distribution of bacteriocin-producing bacteria
in the rumen. Our results demonstrated that bovicin HC5 and bovicin 255 are frequently found among the ruminal
streptococci. Further studies should address the ecological role of these peptides in the rumen ecosystem.
Acknowledgements
CNPq, CAPES, FAPEMIG
References
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