Annals of Microbiology (2019) 69:131–138 https://doi.org/10.1007/s13213-018-1407-2 ORIGINAL ARTICLE Antibacterial activity and lantibiotic post-translational modification genes in Streptococcus spp. isolated from ruminal fluid Yasmin Neves Vieira Sabino1 & Romário Costa Fochat1 & Junior Cesar Fernandes Lima2 & Marlice Teixeira Ribeiro2 & Pedro Braga Arcuri2 & Jailton da Costa Carneiro2 & Marco Antônio Machado2 & Daniele Ribeiro de Lima Reis2 & Alessandra Barbosa Ferreira Machado3 & Humberto Moreira Húngaro1 & João Batista Ribeiro2 & Aline Dias Paiva3 Received: 17 May 2018 /Accepted: 11 November 2018 /Published online: 6 December 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract The production of bacteriocins is frequently described in high microbial diversity environments. The aims of this study were to screen Streptococcus spp. isolated from rumen for their antibacterial potential and to determine the presence of post-translational modification genes for lantibiotic class of bacteriocins. The isolates were tested for production of antibacterial compounds by the spot-on-lawn assay. Presence of interfering factors and the sensitivity to proteinase K were evaluated. The ruminal bacteria were identified by 16S rRNA gene sequencing and the subspecific discrimination of the isolates belonging to the same specie was performed by PFGE. The presence of lantibiotic post-translational modification genes (lanB, lanC,andlanM)intobacterial genomes was performed by PCR. The bacteriocin-like inhibitory substances showed broad inhibitory activity and the producer cells were identified as S. equinus, S. lutetiensis,andS. gallolyticus. According to PFGE, the isolates identified as S. equinus belong to different strains. Three ruminal isolates showed at least one of the lantibiotic post-translational modification genes, and lanC was more frequently detected (75%). The production of broad-spectrum bacteriocin-like inhibitory substances by rumen strains suggests that antimicrobial peptides may play an important role in competition in the complex ruminal ecosystem. Keywords Bacteriocin . Rumen . Streptococcus sp. Antibacterial activity . Lantibiotic . lanC Introduction Bacteriocins comprise a heterogeneous group of peptides based on size, molecular weight, structure, biochemical proper- Antimicrobial peptides are produced by several organisms, ties, thermostability, mechanism of action, presence of post- including plants, insects, mammals, and microorganisms (Li translational modified amino acid residues, and spectrum of in- et al. 2012). Antimicrobial peptides ribosomally synthesized hibitory activity (Kaškonienė et al. 2017). According to Alvarez- by bacteria are known as bacteriocins (Cotter et al. 2005), Sieiro et al. (2016), bacteriocins produced by lactic acid bacteria while antimicrobial peptides that were not fully characterized or other microorganisms can be classified into three main groups, regarding the amino acid sequence and biochemical properties based on the biosynthesis mechanism and biological activity: are defined as bacteriocin-like inhibitory substances (BLIS) (Settani and Corsetti 2008). Bacteriocins and BLIS act as an- – Class I: represented by ribosomally produced and post- tagonistic substances and show bactericidal or bacteriostatic translationally modified peptides (molecular weight < activity against target cells (Cleveland et al. 2001). 10 kDa). This class encompasses all the peptides that undergo enzymatic modification during biosynthesis, which provides molecules with uncommon amino acids * Aline Dias Paiva and structures that have an impact on their properties [email protected] (e.g., lanthionine, heterocycles, head-to-tail cyclization, glycosylation); 1 Faculdade de Farmácia, Universidade Federal de Juiz de Fora, Juiz – Class II: includes unmodified peptides with molecular de Fora, Minas Gerais, Brazil weight lower than 10 kDa, which do not require en- 2 Embrapa Gado de Leite, Embrapa, Juiz de Fora, Minas Gerais, Brazil zymes for their maturation other than a leader pepti- 3 Instituto de Ciências Biológicas e Naturais, Universidade Federal do dase and/or a transporter; Triângulo Mineiro, Uberaba, Minas Gerais, Brazil 132 Ann Microbiol (2019) 69:131–138 – Class III: comprises unmodified peptides with a molecu- 85% N2, 10% H2,and5%CO2). Bacterial growth was deter- lar weight greater than 10 kDa that show bacteriolytic or mined by measuring the optical density at 600 nm. non-lytic mechanism of action. Antagonistic activity and exclusion of interfering In complex ecosystems, with high microbial diversity, as factors the ruminal environment, the production of bacteriocins oc- curs at high frequency (20–50%) (Mantovani et al. 2001; Antagonistic activity was evaluated by the spot-on-lawn Whitford et al. 2001), a feature that can be considered a com- method (Booth et al. 1977). The selected ruminal bacteria petitive advantage for the producer cells. Bacteria belonging were spotted onto BHI agar and incubated anaerobically to the genus Streptococcus, Enterococcus, Ruminococcus,and (37 °C, 18 h). Molten semi-solid BHI (0.75% agar), previous- Butyrivibrio have been characterized as the main bacteriocin ly inoculated with the indicator bacteria (105 CFU mL−1), was producers among ruminal strains (Whitford et al. 2001; poured over agar plates already containing the bacterial colo- Kalmokoff et al. 2003;Wangetal.2012; Chakchouk- nies. Plates were incubated at a suitable temperature and at- Mtibaa et al. 2014). mosphere for each indicator organism. The antimicrobial ac- Bacteriocins naturally produced by ruminal bacteria have tivity was determined by the presence of inhibition zones of shown an increased stability in the rumen, mostly because they the indicator organism growth around the colonies of selected had evolved in the same competitive environment, as well as a bacteria (> 6 mm diameter). As bacteriocins and BLIS are spectrum of activity similar to nisin, the most widely studied usually active against phylogenetically related bacteria or bacteriocin (Russell and Mantovani 2002). Thus, bacteriocins against bacteria belonging to the same ecological niche produced by ruminal isolates could be used in order to modify (Cotter et al. 2005), we selected Streptococcus macedonicus the microbial populations in the rumen and to induce changes and Streptococcus equinus, strains also isolated from rumen in ruminal fermentation parameters, similarly to ionophores and belonging to the same culture collection, to be used as (Mantovani and Russell 2002; Kisidayová et al. 2009). In ad- indicator bacteria. dition, the application of bacteriocins or even the bacteriocin- Ruminal bacterial isolates with proven antagonistic activity producing bacteria has been suggested in areas such as food were tested for the presence of interfering factors, in order to and pharmaceutical industry, odontology, agriculture, and hu- verify if the antagonistic activity observed was indeed due to man and veterinary medicine (Drider et al. 2016; Barbosa et al. the production of antimicrobial peptides and not to the activity 2017;Kaškonienė et al. 2017;Lietal.2017). of other factors, such as bacteriophage infection or production In the present study, we have evaluated the production of of acidic compounds. bacteriocin-like inhibitory substances by ruminal bacteria, as To evaluate the presence of bacteriophage, an agar piece well as detected several genes involved in post-translational (3 mm) of the inhibition zone observed in the spot-on-lawn modification of lantibiotics, a commonly reported class of method was aseptically removed, macerated in saline (NaCl bacteriocins. 0.9%, pH 7.0), and centrifuged (12,000g, 20 min). Then, an aliquot of 200 μL of the supernatant was transferred to 3 mL culture previously inoculated with the indicator microorgan- Materials and methods ism. After incubation for 10 min at room temperature, an aliquot of 200 μL was added to 3.5 mL of semi-solid BHI, Microorganisms and growth conditions which was poured over a prior layer of solid BHI (Turner and Jordan 1981). After incubation, the plates were visually Thirty Gram-positive bacteria, previously isolated from bo- inspected for the presence of lytic zones, indicative of bacte- vine ruminal fluid, and belonging to the culture collection of riophage action. the Laboratory of Rumen Microbiology, Embrapa Dairy To exclude the possibility of the production of acidic com- Cattle, Juiz de Fora, MG, Brazil, were randomly selected for pounds being responsible for the observed antagonism, after in vitro screening of antagonistic activity. The isolates were the spot-on-lawn test, the pH inside and outside of the growth maintained in glycerol containing brain heart infusion (BHI) inhibition zones was determined twice using pH test strips broth (10% final concentration), at − 80 °C, until use. (Advantec Narrow Range pH Test papers, from 5.4 to 7.0 pH). The selected bacteria were grown in BHI broth, under an- aerobic conditions, at 39 °C, overnight. The culture media were Effect of proteinase K on the biological activity prepared anaerobically, under O2-free carbon dioxide flux, and of the antagonistic substances the final pH was adjusted to 6.5. Liquid media were distributed in tubes, under O2-free carbon dioxide flux, tightly closed with In order to evaluate the protein nature of antagonistic sub- rubber lids and sealed with an aluminum seal; solid media were stances detected on the spot-on-lawn method, sensitivity to distributed on