Kim et al. Journal of Animal Science and Biotechnology (2021) 12:93 https://doi.org/10.1186/s40104-021-00614-x RESEARCH Open Access Specific inhibition of Streptococcus bovis by endolysin LyJH307 supplementation shifts the rumen microbiota and metabolic pathways related to carbohydrate metabolism Hanbeen Kim1, Tansol Park2, Inhyuk Kwon3 and Jakyeom Seo1* Abstract Background: Endolysins, the bacteriophage-originated peptidoglycan hydrolases, are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance. The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S. bovis and to explore changes in rumen fermentation and microbiota in an in vitro system. Two treatments were used: 1) control, corn grain without LyJH307; and 2) LyJH307, corn grain with LyJH307 (4 U/mL). An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers (450 ± 30 kg) and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time. In vitro dry matter digestibility, pH, volatile fatty acids, and lactate concentration were estimated at 12 h, and the gas production was measured at 6, 9, and 12 h. The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing. Results: LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S. bovis (approximately 70% compared to control, P = 0.0289) and increased ruminal pH (P = 0.0335) at the 12 h incubation. The acetate proportion (P = 0.0362) was significantly increased after LyJH307 addition, whereas propionate (P = 0.0379) was decreased. LyJH307 supplementation increased D-lactate (P = 0.0340) without any change in L-lactate concentration (P > 0.10). There were no significant differences in Shannon’s index, Simpson’s index, Chao1 estimates, and evenness (P > 0.10). Based on Bray-Curtis dissimilarity matrices, the LyJH307 affected the overall shift in microbiota (P = 0.097). LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium, WCHB1–41, unclassified genus Selenomonadaceae, Paraprevotella, vadinBE97, Ruminococcus gauvreauii group, Lactobacillus, Anaerorhabdus furcosa group, Victivallaceae, Desulfuromonadaceae, and Sediminispirochaeta. The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism. * Correspondence: [email protected] 1Department of Animal Science, Life and Industry Convergence Research Institute, Pusan National University, 1268-50 Samrangjin-ro, Miryang 50463, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. Journal of Animal Science and Biotechnology (2021) 12:93 Page 2 of 15 Conclusions: LyJH307 caused a reduction of S. bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism. This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota. Keywords: Endolysin, Rumen microbiota, Ruminal acidosis, Streptococcus bovis Background The CBD affects host specificity by recognizing and binding The use of high amounts of non-fiber carbohydrate to the host PG layer, and the EAD can degrade the PG layer (NFC) is generally accepted by farmers to meet the nu- when increased endolysin-PG proximity is made by CBD tritional energy requirements of high-performance cattle; [14]. The number of studies related to endolysin develop- however, this strategy often carries nutritional disorders, ment has been improved by the emergence of multi-drug including the occurrence of ruminal acidosis. Generally, resistant bacteria, which render many types of antibiotics ruminal acidosis can be divided into two types, subacute ineffective. Many researchers have shown that endolysin ruminal acidosis (SARA) and acute acidosis [1]. The out- can lyse gram-positive bacteria when endolysin is adminis- break of ruminal acidosis starts from increased inflow of tered externally because of the absence of an outer NFC into the rumen, and it can stimulate the overgrowth membrane such as a lipopolysaccharide layer [15–17]. of amylolytic bacteria, which induce the increase of vola- Therefore, endolysin is a promising replacement for antibi- tile fatty acids (VFA) and lactate production and decrease otics. In the food and biomedical health fields, many endo- ruminal pH [2]. In this condition, excessive production of lysin applications have tried to solve the problem of VFA with a concomitant increase of lactate fermenting antibiotic resistance [18]; however, in the livestock science bacteria produces SARA; however, improper growth of field, to the best of our knowledge, limited trials have been lactate fermenting bacteria can induce acute acidosis by reported on endolysin development for treating mastitis- the accumulation of lactate in the rumen [1, 3]. Both types associated bacteria, specifically Staphylococcus aureus [19], of acidosis can cause a decrease in production perfor- and infectious bacteria on fresh cheese, specifically Listeria mances (average daily gain, growth rate, and milk yield) monocytogenes [20]. Moreover, there have been no attempts and create an abnormal balance of rumen microbiota, to apply endolysin for modulating ruminal metabolic dis- causing significant economic damage to farmers [4]. ease via the specific control of ruminal bacteria in the Streptococcus bovis is one of the main ruminal amylo- rumen. lytic bacteria that can produce lactate as the major fer- In a previous study, we developed endolysin LyJH307 mentation product under low ruminal pH conditions. with high lytic activity against S. bovis [15]. Endolysin Under a high grain diet, S. bovis can generate more ATP LyJH307 has a distinct combination of conserved do- per hour than other ruminal amylolytic bacteria, and S. main consisting of N-terminal NlpC/P60 superfamily bovis features not only high acid resistance, but also (EAD) and C-terminal Zoocin A target recognition do- rapid growth rate [5, 6]. Therefore, S. bovis is one of the main superfamily (CBD), and the endolysin LyJH307 had critical initiators of ruminal acidosis. potent lytic activity in a variety of condition (pH, 5.0– Great efforts have been undertaken to prevent an out- 6.0; temperature, 25–55 °C; NaCl concentration, 0–250 break of ruminal acidosis, such as a change of feeding mmol/L). Moreover, it confirmed that endolysin strategies (e.g., a gradual increase of concentration LyJH307 retained lytic activity against the S. bovis in sin- source) [7], use of buffering agent [8], or feeding rumen gle culture media. Therefore, we expected that endolysin modulators such as chemical agent (α-amylase and glu- LyJH307 would have potent lytic activity against the S. cosidase inhibitor) [9]. In addition, there have been a bovis group in rumen condition. few studies attempting to inhibit S. bovis specifically by Therefore, the objectives of the present study were to immunization [10, 11] and bacteriocin [12]. However, to (1) investigate the lytic activity of endolysin LyJH307 the best of our knowledge, there have been no studies against S. bovis in an in vitro system, and (2) explore the attempting to modulate ruminal acidosis by endolysin. changes in rumen fermentation and microbiota. This Endolysin is bacteriophage-originated peptidoglycan (PG) study hypothesized that the endolysin LyJH307 might in- hydrolases that can break down the PG layer of the host duce the reduction of S. bovis in an in vitro system, from within the bacteria during the final stage of the repli- thereby increasing ruminal pH. cation cycle [13]. In general, endolysin targeting gram- positive bacteria comprises at least two conserved func- Methods tional domains, N-terminal enzymatically active domain The protocols for this study concerning animal use were (EAD) and C-terminal cell wall binding domain (CBD). reviewed and approved by the Animal Research Ethics Kim et al. Journal of Animal Science and Biotechnology (2021) 12:93 Page 3 of 15 Committee of Pusan National University (Pusan, Korea, amylase) was used to estimate aNDF and was expressed PNU-2020-2827). inclusive of residual ash. The NFC of the experimental diet was estimated as follows: NFC = [100 – ash – EE – Preparation of recombinant endolysin LyJH307