Microbial Shifts in the Swine Distal Gut in Response to the Treatment with Antimicrobial Growth Promoter, Tylosin
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Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter, tylosin Hyeun Bum Kima, Klaudyna Borewicza, Bryan A. Whiteb, Randall S. Singera, Srinand Sreevatsana, Zheng Jin Tuc, and Richard E. Isaacsona,1 aDepartment of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108; bDepartment of Animal Sciences, University of Illinois, Urbana, IL 61801; and cMinnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN 55455 Edited* by Harley W. Moon, Veterinary Medical Research Institute, Ames, IA, and approved August 13, 2012 (received for review March 27, 2012) Antimicrobials have been used extensively as growth promoters otherwise be used by the host. Therefore, it is highly likely that (AGPs) in agricultural animal production. However, the specific growth promotion could be mediated by selection of specific mechanism of action for AGPs has not yet been determined. The bacterial populations that contribute to the metabolism of the work presented here was to determine and characterize the animal thereby enhancing feed conversion. microbiome of pigs receiving one AGP, tylosin, compared with It is our hypothesis that AGPs cause alterations of the gut untreated pigs. We hypothesized that AGPs exerted their growth microflora, and these changes bring increased feed efficiency to promoting effect by altering gut microbial population composi- animals resulting in growth promotion. A first step to un- tion. We determined the fecal microbiome of pigs receiving tylosin derstanding the process of growth promotion is to identify and compared with untreated pigs using pyrosequencing of 16S rRNA describe alterations in the gut microflora of the pig. Recently gene libraries. The data showed microbial population shifts there have been three reports on microbial shifts in response to representing both microbial succession and changes in response the use of AGPs using culture independent assessments (11–13). to the use of tylosin. Quantitative and qualitative analyses of Overall, these studies were small in scale and used animals held sequences showed that tylosin caused microbial population shifts in infectious disease isolation facilities and did not replicate in both abundant and less abundant species. Our results estab- conditions comparable to how commercial pigs are reared. The lished a baseline upon which mechanisms of AGPs in regulation of work presented here was designed to better understand the health and growth of animals can be investigated. Furthermore, effects that a single growth promoter had on the microbiome of the data will aid in the identification of alternative strategies to pig feces using a longitudinal study design that used pigs in improve animal health and consequently production. typical commercial settings. Tylosin, a member of the macrolide family of antimicrobials, was selected for this study because it is intestinal microbiota | microbiome maturation | metagenomics thought to be the most commonly used AGP. The results pre- sented here demonstrate that tylosin produces consistent and fi fl n the 1940s, it was observed that animals fed dried mycelia of speci c alterations of the distal intestinal micro ora of the pig. Streptomyces aureofaciens that naturally contained chlortetra- I Results MICROBIOLOGY cycline exhibited enhanced growth (1). Since the 1950s, anti- microbials have been used in agricultural animal production for DNA Sequence Data and Quality Control. The studies described the treatment, control, and prevention of infectious diseases, as were designed to detect changes in the fecal microbiome of pigs well as for growth promotion (2, 3). The National Swine Survey being raised in conventional production units over time. The showed that 41% of feeds included one or more individual study design used two groups of pigs (10 pigs per group). One antimicrobials and 19% included combinations of antimicrobials. group received tylosin in their feed during the entire experi- Tylosin and bacitracin were most often used in feeds for grower mental period, whereas the second group did not receive tylosin. and finisher pigs (4, 5). Because the use of antimicrobials leads to The protocol was performed using two independent farms. DNA the emergence of resistance to antimicrobials (6, 7), efforts to sequences in each group and farm were analyzed as pooled reduce their use with the goal of preserving the efficacy of cur- groups based on our previous results that demonstrated strong rent antimicrobials are being implemented. similarities between the fecal microbiomes of pigs within the The specific mechanisms whereby antimicrobials act as growth same pen (14). However, because samples from each pig had a bar code identifier, we were able to quantify the number of promoters (AGPs) are unclear. Early studies demonstrated that sequence reads per animal. A total of 504,204 and 1,543,479 oral antimicrobials did not have growth-promoting effects when DNA sequence reads were generated from farm 1 and farm 2, given to germ-free animals (8). Therefore, it is likely that growth respectively. Over 86% and 94% of the total number of sequence promotion is based on changes to the gut microbiota. Several reads from farm 1 and farm 2 passed the quality control imple- mechanisms of how growth promoters act have been postulated mented in this study, resulting in 435,225 and 1,445,371 including the prevention of subclinical infections and the re- sequences for farm 1 and farm 2. The total number of sequence duction in microbial use of nutrients (2, 9). AGPs also could act reads from farm 2 was 3.3 times larger than that of farm 1 be- by reducing the presence of opportunistic pathogens in animals cause of the improved sequencing chemistry used with samples fed AGPs. Increased immune mediators such as interleukin-1 induced by gut bacteria have been shown to reduce feed con- version in animals with a conventional microflora (10), which ’ fl Author contributions: H.B.K., B.A.W., R.S.S., S.S., and R.E.I. designed research; H.B.K., K.B., illustrate that the host s response to the indigenous micro ora and R.E.I. performed research; H.B.K. contributed new reagents/analytic tools; H.B.K., could be a factor limiting growth efficiency. Direct effects of B.A.W., R.S.S., S.S., Z.J.T., and R.E.I. analyzed data; and H.B.K. and R.E.I. wrote the paper. AGPs on the gut microflora might result in decreased competi- The authors declare no conflict of interest. tion for nutrients and a reduction in microbial metabolites that *This Direct Submission article had a prearranged editor. depress animal growth (9). Certain bacteria in the gut are known Data deposition: The sequences used in this paper are publicly available at Galaxy, https:// to be metabolically important for animal growth. For example, main.g2.bx.psu.edu/u/kim-et-al/h/kim-et-al. cellulolytic bacteria in ruminants digest cellulose into ferment- 1To whom correspondence should be addressed. E-mail: [email protected]. able glucose that serves as a substrate for further microbial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fermentation and for use by the animal. Cellulose would not 1073/pnas.1205147109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1205147109 PNAS | September 18, 2012 | vol. 109 | no. 38 | 15485–15490 Downloaded by guest on October 2, 2021 from farm 2 (GS-FLX chemistry for farm 1 and titanium composition of the fecal microbiome between animals receiving chemistry for farm 2). The average number of sequence reads tylosin and those that did not receive tylosin and how it changed generated per pig was 4,352 for farm 1 and 14,454 for farm 2. over time (16). The results of phylum and class distributions are The median sequence read length for both the tylosin and no- shown in Fig. 1. Two phyla, Firmicutes and Bacteroidetes, were tylosin groups on farm 1 and farm 2 was 137–138 bases with no the most dominant in the fecal samples regardless of age of pigs ambiguous bases. The range of sequence reads was 81–196 bases or treatment group, and comprised more than 92% of the total and, when a homopolymer run was detected, the median length sequences. The proportion of sequences that could not be of the homopolymer was 5 bases (Table S1). assigned to a phylum using RDP classifier ranged from 1.61% to 6.37%. Bacteria in the phylum Spirochaetes increased over time Microbial Diversity. The diversity of microbial communities using ranging from 0.07% to 1.85%. The other phyla represented less pooled sequence reads from all ten pigs in each group was than 1% of the total at all time points. The proportion of bac- measured using Shannon–Weaver and Simpson (1-D) diversity teria in the phyla Firmicutes, Spirochaetes, and unclassified indices (15). The diversity indices used represent how many dif- phyla increased as the pigs aged, whereas the proportion of ferent taxa are present in a sample, and higher numbers indicate bacteria in the phyla Bacteroidetes, Actinobacteria, and Pro- higher diversity. The average Shannon–Weaver and Simpson (1- teobacteria decreased. These changes were consistently observed D) index values showed highly diverse microbial communities in both farms. When the relative abundance of bacteria at the with mean values per group of 4.87 and 0.96 for farm 1, and 5.28 phylum level was compared between tylosin and no-tylosin and 0.97 for farm 2. The range of these calculated values over the groups of the same age, there were no significant differences five sampling times (3-wk intervals starting at 10 wk of age and (Fig. 1 A and B). ending 22 wk of age) was 4.39–5.50 for Shannon–Weaver and Bacteria in the classes Clostridia, Erysipelotrichi, Bacilli, 0.95–0.98 for Simpson (1-D).