Academic supervisors
Senior researcher, Nuria Canibe, PhD
Department of Animal Science, Faculty of Science and Technology,
Aarhus University, 8830, Tjele, Denmark.
Senior researcher Emeritus, Bent Borg Jensen, PhD
Department of Animal Science, Faculty of Science and Technology,
Aarhus University, 8830, Tjele, Denmark.
Assessment committee
Associated Professor Jan Værum Nørregaard, PhD (Chair)
Department of Animal Science, Faculty of Science and Technology,
Aarhus University, Tjele, Denmark.
Professor Jan Erik Lindberg, PhD
Department of Animal Nutrition and Management,
Swedish University of Agricultural Sciences, Uppsala, Sweden.
Professor Ulrike Weiler, PhD
Institute of Animal Husbandry and Animal Breeding,
Behavioral Physiology of Farm Animals,
University of Hohenheim, Stuttgart, Germany
I Preface
This PhD thesis is submitted to the Faculty of Science and Technology, Aarhus University, Denmark, to fulfil the requirements of the PhD degree. It represents my work conducted at Department of Animal Science, in the research group of Immunology and Microbiology, from June 2013 to November 2016, interrupted by a maternity leave, giving in a total period of 3.5 years. This study has been supervised by the main supervisor, senior researcher Nuria Canibe, and the co-supervisor, senior researcher emeritus Bent Borg Jensen.
The work was carried under the project ‘Organic pig production without castration’ that is part of the Organic RDD program, which is coordinated by International Centre for Research in Organic Food Systems, ICROFS. It was founded by Danish AgriFish Agency, Ministry of Food, Agriculture and Fisheries (Project 3405-10-OP-00134). The PhD student was supported by a scholarship from the China Scholarship Council (CSC).
II Acknowledgments
Foremost, I would like to express my sincere appreciation and thanks to both of my supervisors Nuria Canibe and Bent Borg Jensen for their continuous support of my study and research, for their patience, motivation, enthusiasm, and unparalleled knowledge. Without their consistent guidance, it would not be possible to complete this project. I would also like to thank my master supervisor Jiufeng Wang who recommended me to join this group.
Besides my supervisors, I would like to thank all my co-authors for their insightful comments and contributions to my papers. I must say a special thank you to Dr Ole Højberg and Dr Samantha Joan Noel, for their guidance, support and encouragment.
My sincere thanks also goes to all technicians: Mona Dinsen, Karin Durup, Trine Poulsen, Thomas Rebsdorf, Helle Handll, Kasper Poulsen, Britta Poulsen for skillful technical assistance in carrying out my experiments. I also appreciate help from Henry Johannes Høgh Jørgensen for assisting to perform the catheter surgery.
I appreciate my previous and present officemates: Morten Poulsen, Grethe Venås Jakobsen, Zhigang Zhu, joyfully accompany and support. A warm thanks to all my colleagues in the group of Immunology and Microbiology for creating a harmonious environment. In particular, I would like to thank Ann-Sofie Riis Poulsen and Marlene Fredborg, for their willingness to share and help. And a big thanks to our secretory Mette Graves Madsen for all the assistance.
Many thanks go to Nørresø kollegiet residents, the Chinese community in Foulum and the International club. Without them, my life in Denmark would not be that colorful. I would also like to thank all of my friends for supporting and understanding.
Last but not the least, I am thankful to my family: my selfless parents for being babysitting in Denmark, my talented sister for backing, and my dear husband Jin for supporting me spiritually throughout my PhD and my life in general. In particular, I am grateful to my little daughter Shirley for reminding me that I am a mother besides a PhD.
Xiaoqiong Li/
Foulum, December 2016
III List of scientific publications and manuscripts
Paper I: Li X, Jensen RL, Højberg O, Canibe N, Jensen BB. 2015. Olsenella scatoligenes sp. nov., a 3-methylindole- (skatole) and 4-methylphenol- (p-cresol) producing bacterium isolated from pig faeces. International Journal of Systematic and Evolutionary Microbiology 65:1227–33.
Paper II: Li X, Højberg O, Noel SJ, Canibe N, Jensen BB. 2016. Draft Genome Sequence of Olsenella scatoligenes SK9K4T, a Producer of 3-Methylindole (Skatole) and 4-Methylphenol (p-Cresol), Isolated from Pig Feces. Genome Announcements 4:e00042-16.
Paper III: Li X, Marshall IPG, Schreiber L, Højberg O, Canibe N, Jensen BB. 2016. Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole. Genome Announcements 4:e00007-16.
Paper IV: Li X, Jensen BB, Højberg O, Noel SJ, Canibe N. Development of a species-specific Taqman-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet (planned to be submitted to Applied Microbiology Biotechnology)
Paper V: Li X, Højberg O, Canibe N, Jensen BB. 2016. Phylogenetic diversity of cultivable butyrate-producing bacteria from pig gut content and feces. Journal of Animal Science 94:377–381.
Paper VI: Li X, Jensen BB, Canibe N. Effect of chicory roots and exogenous butyrate on skatole production and gut microbiota of entire male pigs (planned to be submitted to Applied and Environmental Microbiology).
IV List of abbreviations
CYP Cytochromes P450
DF Dietary fiber
DGGE Denaturing gradient gel electrophoresis
DP Degree of polymerization
FISH Fluorescence in situ hybridization
FOS Fructo-oligosaccharides
GI Gastrointestinal
GnRH Hypothalamic gonadotropin-releasing hormone
HTS High-throughput sequencing
IAA Indole acetic acid
IPA Indole propionic acid
ITF Inulin-type fructans
MGB Minor groove binder
NGS Next-generation sequencing
NSP Non-starch polysaccharides
OTU Operational taxonomic unit
QIIME Quantitative Insights Into Microbial Ecology qPCR Quantitative real-time PCR
RPS Raw potato starch
RS Resistant starch
SCFA Short-chain fatty acid
TRP L-tryptophan
T-RFLP Terminal restriction fragment length polymorphism
WGS Whole-genome shotgun
V Summary
Skatole, a cytotoxic and malodorous compound, is the main causer of boar taint, which is an offensive odor and flavor released upon heating meat from some pubertal or sexually mature male pigs. Due to animal welfare concerns, there is an increasing desire to stop surgical castration within the European Union by 2018, turning boar taint into a challenge in pig production. Skatole is produced by microbial fermentation of L-tryptophan (TRP) in the hindgut. The production involves a two-step process, in which the precursor TRP is first deaminated to indole acetic acid (IAA), which is then further decarboxylated to skatole. Difficulties arise in controlling boar taint, due to the lack of knowledge on the specialized gastrointestinal skatole-producing bacteria. Skatole levels can be reduced by feeding certain dietary fibers (DF), making this strategy an alternative to surgical castration. Dietary fiber sources rich in inulin-type fructans (e.g. chicory roots and Jerusalem artichoke) are the most effective in reducing skatole formation as seen so far. However, the exact mechanism underlying the skatole reducing effect of DF remains unclear.
Hence, the objective of this PhD thesis was to screen and characterize the skatole-producing bacteria inhabiting the hindgut of pigs, their metabolism, genome, quantification, and to elucidate the mode of action behind the reducing effect of DF on skatole production in the hindgut of pigs.
We screened 122 OTUs, which represent 2678 isolates from the GI-tract of pigs for their ability to produce skatole. Results showed that only one strain, O. scatoligenes SK9K4, had the capacity to produce skatole from IAA, but not from TRP. The 2.47 Mbp draft genome sequence of O. scatoligenes SK9K4T confirmed that it possesses the tyrosine degradation IV (to p-cresol) pathway, and offered the possibility to identify candidate genes for skatole production by comparative genomic analysis. Use of Taqman-MGB qPCR, enabled specific enumeration of O. scatoligenes, and showed that this bacterium only accounted for less than 0.01% of the total bacterial population in the pig hindgut.
High levels of dietary chicory roots promoted O. scatoligenes proliferation, and selectively promoted the growth of specific butyrate-producing bacteria such as Megasphaera elsdenii, but not a general increase in the population of butyrogenic bacteria in pig gut. This indicated that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of the skatole-producing bacteria or by a general increasing of the butyrate-producing bacteria. Intracecal infusion of butyrate alone did failed to reduce skatole formation in the pig hindgut.
In conclusion, cultivable skatole-producing bacteria in the pig GI-tract seem to be limited to one
VI species, i.e., O. scatoligenes. The known effect of chicory roots for reducing skatole production is neither by inhibiting the growth of this skatole-producing bacteria in the hindgut, nor by inhibition of cell apoptosis by butyrate, but is more likely due to more TRP incorporated into microbial biomass.
VII Resumé (Danish summary)
Skatol, en cytotoksisk og ildelugtende forbindelse, er den vigtigste komponent af ornelugt, en ubehagelig lugt og smag der frigives ved opvarmning af kød fra specielt seksuelt modne hangrise. Kirurgisk kastration af hangrise er en almindelig praksis i mange lande for at forhindre ornelugt. Kirurgisk kastration er imidlertid smertefuld og dyrevelfærdsmæssigt problematisk. Der er derfor i Den Europæiske Union indgået en frivillig aftale om at ophøre med at kastrere grise kirurgisk fra den 1. januar 2018. Skatol er et mikrobielt nedbrydningsprodukt der dannes ved mikrobiel fermentering af aminosyren L-tryptophan (TRP) i blind- og tyktarmen. Det produceres i en to-trins proces i hvilken TRP først deamineres til IAA der efterfølgende decarboxyleres til skatol. Det forhold, at man ikke kender den eller de bakterier der producerer skatol i blind- og tyktarmen, gør det vanskeligt at kontrollere skatole produktionen. Man ved, at tilsætning af visse kostfibre (DF) til fodret reducerer produktionen af skatol i blind- og tyktarmen, hvilket gør denne strategi til et alternativ til kirurgisk kastration. Inulinholdige kostfiberkilder som cikorierødder og jordskokker has vist sig at være de mest effektive til at reducere produktionen af skatol, men den nøjagtige mekanisme der ligger til grund for denne effekt er stadig uklar.
Formålet med nærværende ph.d.-afhandling var derfor at screene og karakterisere de skatol producerende bakterier fra blind- og tyktarmen hos svin, at kortlægge deres stofskifte og genomer, at kvantificere deres forekomsten i blind- og tyktarmen og at belyse den virkningsmekanismen der ligger bag den reducerende effekt af DF på skatol produktion i blind- og tyktarmen hos svin.
Et hundrede og toogtyve OTUs, repræsenterende 2678 isolater fra mave-tarmkanalen hos grise blev screenet for deres evne til at producere skatol. Resultaterne viste, at kun én stamme, O. scatoligenes SK9K4, var i stand til at producere skatol. O. scatologenes kan producere skatol fra IAA, men ikke direkte fra TRP. Draft genom sekventering af O. scatoligenes SK9K4T genomet (2,47 Mbp) bekræftede at O. scatoligenes besidder generne for tyrosin nedbrydning til p-cresol, og vil gøre det muligt at identificere kandidatgener for skatol produktion ved komparativ genom analyser. Brug af Taqman-MGB qPCR, gjorde det muligt at kvantificere populationen af O. scatoligenes i blind og tyktarmen. Resultaterne viste, at bakterien udgør mindre end 0,01 % af den samlede bakterie population i grises blind- og tyktarm.
Tilsætning af cikorierøder til fodret øgede forekomsten af O. scatoligenes i blind- og tyktarmen og væksten af specifikke smørsyre producerende bakterier som Megasphaera elsdenii, men bevirkede ikke
VIII en generel stigning i populationen af smørsyreproducerende bakterier. Dette indikerede, at den kendte virkning af cikorierødder på produktionen af skatol ikke sker ved en hæmning af væksten af de skatol-producerende bakterier eller ved en generel forøgelse af de smørsyreproducerende bakterier. Infusion af smørsyre i blindtarmen var ikke i stand til at reducere produktionen af skatol i blind- og tyktarmen.
Sammenfattende kan der konkluderes at produktionen af skatol i grises mave- tarmkanal syntes at være begrænset til én art: O. scatoligenes. Den kendte virkning af cikorierødders evne til at reducere produktionen af skatol skyldes hverken en reduktion i populationen af skatol producerende bakterier eller en hæmning af apoptose forårsaget af en øget butyrat produktion. Selv om en endelig konklusion ikke kan drages ud fra de opnåede resultater, er virkningen højt sandsynlig nærmere en højere inkorporering af TRP som bakteriel biomasse i tilstedeværelse af fermenterbare DF.
IX Table of contents
Preface ...... II
Acknowledgments ...... III
List of scientific publications and manuscripts ...... IV
List of abbreviations ...... V
Summary ...... VI
Resumé (Danish summary) ...... VIII
Table of contents ...... X
1. General introduction ...... 1
2. Background ...... 3 2.1. Boar taint ...... 3 2.2. Androstenone ...... 4 2.3. Skatole ...... 4 2.3.1. Skatole production in the hindgut ...... 6 2.3.2. Skatole absorption through gut mucosa ...... 8 2.3.3 Skatole metabolism in the liver and kidneys ...... 8 2.3.4. Skatole deposition in adipose tissue ...... 9 2.4. Prevention of boar taint ...... 9 2.4.1. Surgical Castration ...... 9 2.4.2. Genetic selection ...... 10 2.4.3. Gender selection (sperm sexing) ...... 10 2.4.4. Immunocastration ...... 11 2.4.5. Management strategies ...... 11 2.5. Dietary fiber to reduce skatole ...... 12 2.5.1. Dietary fiber ...... 12 2.5.2. Effects of dietary fiber on skatole production ...... 14 2.5.3. Mechanisms for the reducing effect of dietary fiber on skatole production ...... 17
3. Hypothesis and objectives ...... 18
4. Methodological consideration ...... 19
X 4.1. Standards for describing new taxa of bacteria ...... 19 4.2. Techniques for studying the gut microbiome ...... 21 4.2.1. Culture-dependent techniques ...... 21 4.2.2. Quantitative real-time PCR ...... 22 4.2.3. Next-generation sequencing ...... 23 4.3. In vivo experimental design ...... 27
5. Paper I ...... 29 5.1 Supplementary Materials for Paper I ...... 37
6. Paper II ...... 44
7. Paper III ...... 47
8. Paper IV ...... 50
9. Paper V ...... 80 9.1 Supplementary Materials for Paper V ...... 86
10. Paper VI ...... 88
11. General discussion ...... 123 11.1. Skatole-producing bacteria ...... 124 11.2. Quantification of skatole-producing bacteria ...... 125 11.3. Butyrate-producing bacteria ...... 127 11.4. Effect of dietary fiber on skatole production ...... 127 11.5. Effect of dietary fiber on microbial activity ...... 128 11.6. Effect of dietary fiber on skatole-producing bacteria ...... 129 11.7. Effect of dietary fiber on butyrate-producing bacteria ...... 129 11.8. Effect of exogenous butyrate on skatole production ...... 130
12. Conclusions ...... 131
13. Perspectives ...... 132
14. References ...... 134
XI 1. General introduction
Boar taint is an offensive odor and flavor arising during heating of pork from adult entire males, caused primarily by accumulation of skatole and androstenone in fat (Jensen et al. 2014). Skatole is a cytotoxic and malodorous compound produced by microbial fermentation of L-tryptophan (Whitehead et al. 2008). Skatole is hence produced in the hindgut of female, male and castrated pigs. However, boar taint rarely occurs in meat from female or castrated male pigs. If uncastrated, the meat of 2.3 % of Danish entire male pigs is tainted (> 0.25 ppm skatole in the backfat), increasing to 11% when measured by the human nose score method, and 37% have a backfat androstenone level > 1.0 ppm (Maribo et al. 2014). ‘Boar tainted’ meat is disagreeable for most consumers, leading to economic loss in the meat industry.
Currently, surgical castration of male piglets is a common practice worldwide in the pig production to prevent boar taint. However, due to animal welfare concerns, there is an increasing desire to stop such surgical castrations within the European Union by 2018. So far, no single alternative to surgical castration guarantees the complete absence of boar taint while keeping the economic advantages of rearing entire males and avoiding inpaired welfare at the same time (Lundström and Zamaratskaia 2006). The lack of knowledge on various aspects of skatole and androstenone production, and ways to reduce their levels, makes it difficult to apply strategies that reduce boar taint in entire male pigs. An important aspect in elucidating skatole production and metabolism is the identification of skatole-producing bacteria in the gastrointestinal (GI) tract of pigs. However, knowledge on taxonomy and metabolism of the specialized skatole-producing bacteria in the GI-tract is scarce. Several skatole-producing bacteria have been isolated from different environments, but so far, only Clostridium (C.) scatalogenes, isolated from soil, and C. drakei. isolated from sediment, have been fully characterized and made available from culture collections (Whitehead et al. 2008). Up to now, C. scatalogenes, is the only model organism that has been used for the study of skatole production, hindering efforts to control boar taint.
While fat androstenone levels are mostly affected by genetic factors and by the degree of sexual maturity (Grindflek et al. 2010), the fat skatole levels can be reduced by various feeding strategies (Wesoly and Weiler 2012). Studies showed that fermentable dietary fiber (DF), rich in inulin-type fructans (e.g. chicory roots and Jerusalem artichoke ) is the most effective feed component in reducing skatole production so far (Rideout et al. 2004; Hansen et al. 2006; Byrne et al. 2008; Kjos et al. 2010; Øverland et al. 2011; Vhile et al. 2012; Wesoly and Weiler 2012; Zammerini et al. 2012). However, the
1 precise mechanism behind the impact of DF on the levels of skatole in fat is still unclear. Three main underlying mechanisms of the skatole reducing effect of DF have been proposed:
1) an increased butyrate production due to DF fermentation reduces enterocyte apoptosis and, as such, less endogenous tryptophan from cell debris is available for skatole production by the microbiota in the hindgut (Claus et al. 2003); 2) a high content of fermentable DF in the hindgut increases the microbial activity, resulting in more TRP incorporated as bacterial biomass and thereby, leaving less substrate for skatole production (Jensen et al. 1995a; Xu et al. 2002); 3) DF fermentation alters gut microbiota composition by decreasing skatole-producing bacteria and increasing butyrate-producing bacteria.
This project aimed to identify, characterize, and quantify the skatole-producing bacteria inhabiting the hindgut of pigs, and to elucidate the mode of action behind the reducing effect of DF on skatole production in the hindgut of pigs. This may aid finding strategies to reduce skatole formation in the hindgut of pigs and thereby its deposition in fat with the consequent reduction of boar taint.
2 2. Background
2.1. Boar taint
Boar taint is an off-odor and off-flavor meat trait released upon heating of meat from uncastrated sexually mature male pigs (Jensen et al. 2014). The testicular pheromone, androstenone (5α-androst-16-en-3-one) (Patterson 1968), and tryptophan metabolite, skatole (3-methylindole) (Vold 1970), are the two main compounds responsible for boar taint. Both androstenone and skatole levels coincidently increase around the time of puberty (Zamaratskaia et al. 2005b). Indole might intensify the off-flavour in meat and contribute to a minor degree of boar taint perception (García-Regueiro and Diaz 1989). Both boar taint compounds can reach perceptible concentrations, causing consumer rejections when sensed during heating and consumption of pork meat. Acceptable levels of the two main substances differ between consumers and range between 0.5 - 1 µg/g for androstenone and 0.2 – 0.25 µg/g for skatole (Lundström et al. 2009; Zadinová et al. 2016). So far, the unique threshold level for boar taint has not been established, though. A rapid colorimetric method called “skatole equivalent” for online fat skatole analysis is used online in Danish slaughterhouses, yet no online androstenone detection method is available so far (Haugen et al. 2012). Skatole has a higher impact on consumer acceptability compared to androstenone (Matthews et al. 2000; Whittington et al. 2011). Almost every consumer can perceive skatole (Weiler et al. 2000), whereas only 30%-45% of the consumers are sensitive to androstenone (Bekaert et al. 2011). Women are reported to be more sensitive to androstenone that men.
Prevalence of boar taint varies among studies, ranging from 1% to 30% of the pigs measured based on subjective sensory tests by laboratory panels (Xue and Dial 1997). An EU investigation showed that overall approximately 60% and 30% of the entire males had androstenone levels above 0.5 and 1.0 µg/g, respectively; and approximately 15% and 10% had skatole levels above 0.2 and 0.25 µg/g, respectively, but there was a large variation among countries (Walstra et al. 1999). In Denmark, the test results of a slaughter company showed that 2.3 % of entire male pigs are tainted (> 0.25 ppm skatole in the backfat), increasing to 11% by the human nose score, and 37% have an androstenone level > 1.0 ppm (Maribo et al. 2014). Boar taint renders the meat disagreeable for most consumers with subsequent negative economic repercussions for the meat industry. Thus boar taint appears as an important problem in the production of pork meat (Jensen et al. 2014).
3 2.2. Androstenone
Androstenone (5α-androst-16-en-3-one), exhibiting a urine-like odor, is an endogenous pheromone mainly synthesized in the testis of entire male pigs (Claus et al. 1971; Booth 1982). The production of androstenone is controlled by the pituitary luteinizing hormone (LH), which is under stimulatory control of hypothalamic gonadotropin-releasing hormone (GnRH). The biosynthesis of androstenone is low in young pigs and gradually increases during the establishment of puberty (Andresen 1976). After resorption and distribution via the bloodstream, androstenone is enriched in fat tissue due to its lipophilic character. The metabolic pathways of androstenone are summarized in Figure 1 (Zamaratskaia and Squires 2009; Weiler and Wesoly 2012; Zadinová et al. 2016). Androstenone levels depend mostly on maturity stage and genetic factors. Housing, social environment, nutrition, and photoperiod have also some effect on androstenone levels (Bonneau 1982; Claus et al. 1994; Xue and Dial 1997; Sellier et al. 2000; Bonneau 2006; Tajet et al. 2006). Feeding chicory root to male pigs has been shown to increase Cytochromes P450 (CYP450) (3β-HSD) expression and to decrease androstenone accumulation in fat (Rasmussen et al. 2011; Rasmussen et al. 2012).
2.3. Skatole
Skatole (3-methylindole), exhibiting a fecal-like odor, is produced by anaerobic microbial degradation of L-tryptophan (TRP) in the hindgut (Yokoyama and Carlson 1974; Jensen et al. 1995b; Whitehead et al. 2008). Therefore, skatole is found in both female and male pigs. The significance of skatole is not limited to its contribution to boar taint, as it is also an etiological agent of acute bovine pulmonary edemas and emphysema in ruminants (Deslandes et al. 2001), as well as being a putative pulmonary carcinogen (Weems et al. 2009) and a major halitosis causing compound in humans (Codipilly and Kleinberg 2008). Skatole has pneumotoxic effects on several species, including humans, but not pigs (Bray and Kirkland 1990). Moreover, skatole has a broad bacteriostatic effect, including disrupting bacterial biofilm development, potentially inducing bacterial oxidative stress, and may be genotoxic through formation of DNA adducts (Fukuoka et al. 2015). The metabolic pathways of skatole are summarized in Figure 2 (Zamaratskaia and Squires 2009; Wesoly and Weiler 2012; Zadinová et al. 2016).
4
Figure 1 The metabolic pathways of androstenone. Gonadotropin-releasing hormone (GnRH); Luteinizing hormone (LH); cytochrome P450C17 (CYP17A1); cytochrome b5 (CYB5); 3a- and 3β hydroxysteroid dehydrogenase enzymes (3a- and 3β-HSD); Hydroxysteroid sulfotransferases (SULT2A1 and SULT2B1); UDP-glucuronosyltransferase (UGTs).
5
Figure 2 The metabolic pathways of skatole. Cytochrome 2E1 (CYP2E1); P450 2A (CYP2A); phenol sulfotransferase (SULT1A1); uridine-diposphate-glucuronosyltransferase (UDP).
2.3.1. Skatole production in the hindgut
Production of skatole involves a two-step process in which the precursor TRP is first deaminated to indole acetic acid (IAA), which is then further decarboxylated to skatole (Yokoyama and Carlson 1974; Jensen et al. 1995b; Whitehead et al. 2008). The origin of the TRP as substrate for microbial production of skatole can be from either dietary or endogenous protein sources (Claus et al. 1996; Claus and Raab 1999). Dietary supplementation of free TRP above the requirements or increasing amounts of easily digestible protein sources did not increase skatole production in the hindgut, most probably because
6 free TRP is absorbed in the small intestine, and therefore not available for microbial fermentation in the hindgut. Thus, cell debris resulting from apoptosis of intestinal mucosa cells has been proposed to be a major substrate for skatole formation (Claus et al. 1996; Claus and Raab 1999; Jensen et al. 2014). However, diets with low prececal digestibility of TRP, such as blood or bone meal, yeast slurry from breweries, or yellow peas, have been shown to increase skatole production in the hindgut, indicating that the type rather than the amount of dietary protein is important for the production and deposition of skatole (Jensen et al. 2014). Skatole, indole, and indole propionic acid (IPA) are metabolism products of microbial degradation of TRP in the pig hindgut (Figure 3) (Knarreborg et al. 2002).
Figure 3 Schematic diagram of tryptophan metabolism in the pig hindgut. Adapted from (Deslandes et al. 2001).
Whereas a wide range of bacteria species are capable of degrading TRP to indole (Lee and Lee 2010) and IAA (Spaepen et al. 2007), the key precursor of skatole, only a few specialized gut bacteria can catalyze the steps from IAA to skatole (Deslandes et al. 2001). So far, species from Clostridium, Lactobacillus, Rhizobium, Pseudomonas, Prevotella, Actinomyces and Megasphaera have been reported to catalyze the step from IAA to skatole (Deslandes et al. 2001; Attwood et al. 2006). Moreover, C. aminophilum and C. disporicum may also contribute to skatole formation (Li et al. 2009a; Li et al. 2009b). However, to our knowledge, among these, only two strains, C. scatologenes ATCC
7 25775 and C. drakei SL1 DSM 12750, have been fully characterised and are readily available from culture collections. Unlike C. scatalogenes and C. drakei, which produce skatole directly from TRP (Whitehead et al. 2008), bovine rumen Lactobacillus sp. strain 11201 synthesizes skatole by decarboxylating the IAA, but is unable to produce skatole directly from TRP (Yokoyama et al. 1977; Yokoyama and Carlson 1981). It has been estimated that the population of skatole-producing bacteria accounts for less than 0.01% of the total gut microbiota (Jensen and Jensen 1998). In summary, the production of skatole is primarily dependent on the availability of TRP and the activity of skatole-producing bacteria.
2.3.2. Skatole absorption through gut mucosa
Absorption of skatole occurs along the gut mucosa and is transferred to the liver via the portal vein (Claus et al. 1994). The absorption is very rapid with half-life in blood of approximately 1 hour (Agergaard 1998). Total daily absorption rates of skatole have been estimated to be between 365 and 820 µmol skatole, depending on the diet (Knarreborg et al. 2002). The concentrations of skatole in blood and feces along with skatole in backfat are highly correlated within individuals, suggesting that the amount of skatole absorbed is proportional to the amount produced (Claus et al. 1993; Knarreborg et al. 2002). In addition, the correlation between skatole production, absorption, and deposition indicates that a decrease in skatole production will result in reduced skatole deposition in the fat. Intestinal transit time can be affected by diet and therefore might influence the absorption of skatole from gut mucosa (Jensen and Jensen 1998).
2.3.3 Skatole metabolism in the liver and kidneys
Skatole is mainly degraded in the liver with high efficacy. Approximately 87% of the skatole produced in the hindgut is absorbed through the gut wall and transported to the liver, where the majority is degraded, and the remaining 13% is excreted with the feces (Xue and Dial 1997). The presence of the skatole-metabolizing enzyme CYP450 has also been detected in the kidneys, suggesting that the kidneys may also be involved in skatole metabolism (Puccinelli et al. 2011). The hepatic degradation of skatoles can be divided in two distinct phases: oxidative (phase I metabolism), and conjugative (phase II metabolism) (Zamaratskaia and Squires 2009) (Figure 2). The enzymes involved are various cytochrome P450, among which, CYP2E1 and CYP2A have been identified as the major enzymes responsible for the phase I metabolism of skatole (Diaz and Squires 2000; Doran et al. 2002; Wiercinska et al. 2012). Phase I results in the addition of a hydroxyl group, which is then used to attach
8 a conjugate in phase II. The main enzymes of phase 2 metabolism are sulfotransferase (SULT1A1) and uridine-di-phosphate-glucuronosyltransferase (UGT) (Diaz and Squires 2003). Conjugating of skatole makes it more hydrophilic, facilitating its excretion via urine and reducing its adipose tissue absorption (Zamaratskaia and Squires 2009).
High accretion of skatole in fat of entire male pigs is related to low enzyme activity of both CYP2E1 and CYP2A6 (Squires and Lundström 1997; Zamaratskaia et al. 2005c). Entire male pigs have low CYP450 enzymes expression and activity compared to females and castrates (Skaanild and Friis 1999), resulting in reduced skatole metabolic rate in the liver, and consequently, high skatole accumulation in fat (Diaz and Squires 2000; Zamaratskaia et al. 2006). The fact that skatole accumulation is sex-dependent may be due to a possible inhibition of skatole metabolism in the liver by sex steroids along with androstenone (Babol et al. 1999).
2.3.4. Skatole deposition in adipose tissue
The skatole not degraded in the liver is deposited in peripheral tissues. Due to its lipophilicity, most skatole accumulates in the adipose tissue. Skatole concentration in fat increases if plasma skatole level is elevated for a longer period, and drops within days if colonic skatole formation is reduced due to feeding strategies, such as inulin supplementation (Claus et al. 1994). Leaner breeds have, in general, a higher fat turnover and lower skatole/androstenone concentration, suggesting that the turnover rate of adipose tissue may modify skatole/androstenone accumulation in fat (Aluwé et al. 2011). In summary, the amount of skatole stored in adipose tissue depends on the concentration in the peripheral blood, which is influenced by the production in the hindgut and the degradation capacity of the liver (Knarreborg et al. 2002).
2.4. Prevention of boar taint
2.4.1. Surgical Castration
Worldwide, surgical castration is still by far the most used method to reduce the risk of boar taint. Surgical castration removes the source of androstenone production, leading to a dramatic reduction of androstenone levels. Besides, castration prevents aggressive and sexual behavior in male pigs. The reasons for the reduction of skatole levels in castrated pigs are not well understood. It is likely that testicular steroids are important regulators of skatole metabolism (Lundström and Zamaratskaia 2006). In vitro studies have shown that hepatic clearance of skatole is affected by androstenone, through its
9 inhibiting effect on the CYP450 (CYP2E1) enzymes (Doran et al. 2002; Whittington et al. 2004; Zamaratskaia et al. 2007; Chen et al. 2008).
The procedure of castration has been criticized due to pain, discomfort and an increased risk of infection in piglets caused by this practice (Prunier et al. 2006). Additionally, production of entire males is more profitable because of improved feed conversion, and leaner carcasses compared to castrates (Zamaratskaia and Squires 2009). Due to welfare and economic concerns, there is an increasing desire to cease surgical castration within the European Union by 2018. (http://boars2018.com). Today, in Denmark, approx. 95% of the male pigs are castrated (Bee et al. 2015). Therefore, there is an urgent need for feasible alternatives to surgical castration for boar taint prevention. The potential alternatives to surgical castration are genetic selection, gender selection, immunocastration, and production of entire male pigs by management strategies.
2.4.2. Genetic selection
The heritability (h2) for androstenone and skatole ranges from 0.5 to 0.7 and 0.3 to 0.5, respectively in different pig breeds and crosses, and genetic relationships between androstenone and skatole were estimated between 0.3 and 0.4 (Große-Brinkhaus et al. 2015). Due to the high heritability of androstenone, genetic selection to lower androstenone levels seems to be promising and is applied in several breeding programs (Frieden et al. 2014). However, it takes time to select boar-taint-free genetic lines without any adverse consequence on the fertility and growth performance. Furfure work is still necessary to identify the right genetic markers for pigs exhibiting low androstenone levels (Zadinová et al. 2016). Selection against boar taint must be implemented with caution due to uncertain effects on fertility, sexual maturity, average daily gain and meat quality traits (Bee et al. 2015).
2.4.3. Gender selection (sperm sexing)
Sexing of sperm before insemination to produce only female pigs can be an alternative to avoid boar taint as it does not compromise animal welfare. Although it is common in cattle breeding, it may be considered unacceptable from an ethical point of view in pig production. Due to limited sorting speed in relation to a high number of spermatozoa needed to inseminate pigs, sperm sexing is not likely to become available on a commercial scale in the near future (Jensen et al. 2014).
10 2.4.4. Immunocastration
Immunocastration of male pigs is an attractive alternative to surgical castration to reduce boar taint. Moreover, immunocastrated pigs showed reduced sexual and aggressive behavior compared to entire male pigs (Brunius et al. 2011; Brewster and Nevel 2013). Immunocastration is an active immunization method against GnRH, thereby interrupting the HPG-axis, which is responsible for the reproductive function and production of the testicular steroids (androstenone) involved in boar taint. The effect of immunocastration on performance and carcass and meat quality remains unclear. Results vary greatly depending on genetics, feeding, and housing (Millet et al. 2011; Batorek et al. 2012; Aluwé et al. 2013). Only one commercial vaccine, Improvac®, is current available. Improvac® is approved in over 60 countries worldwide and has been in commercial use in the EU since 2009 (Zamaratskaia and Rasmussen 2015). However, it is not allowed by Danish slaughterhouses because of consumer concerns. Immunocastration is expensive, labor intensive for the pig producers, and not accepted in all markets, for sales parties fear negative reactions from consumers. There is no risk for vaccine residues in the meat, but the vaccine also affects humans. There is a risk of adverse effects on the reproductive system for the farmer if self-injection should happen twice (Brewster and Nevel 2013) .
2.4.5. Management strategies
Before boar-taint-free entire male pigs can be produced on a large scale, management practices adapted to rearing entire male pigs (i.e. feeding regimes, housing facilities, etc.) will also be necessary. Improvement of hygiene of the rearing environment, fasting for more than 6 hours before slaughter, minimization of protein sources with low ileal digestibility, and supplementation of easily fermentable DF (see section 2.5) are reasonable strategies to reduce fat skatole. However, there are inconsistent results in the literatures (Jensen et al. 2014). Other feeding choices, such as liquid feeding, or addition of enzymes, antibiotics, bioactive components, adsorbent materials, organic acids, etc are not sufficiently investigated or inappropriate to apply at farm level (Jensen et al. 2014). Results of a recently study suggested that hydrolysable tannins can potentially reduce skatole in entire male pigs (Čandek-Potokar et al. 2015; Bilić-Šobot et al. 2016)
Fat androstenone deposition is only marginally or not affected by feeding regimes (Wesoly and Weiler 2012). It is commonly accepted that increasing slaughter weight is associated with more sexually mature entire pigs and consequently increased androstenone production. Therefore, reducing slaughter weight is likely to reduce the androstenone level, but the effectiveness of this approach depends on
11 breed (Aluwé et al. 2011). Stress and social and physical environment are also likely to affect fat androstenone, but a complex interaction with weight, age, genetics, and nutrition has to be taken into account (Jensen et al. 2014).
In summary, at present, all alternatives have advantages and disadvantages, and no single alternative guarantees the complete absence of boar taint while keeping the economic advantages of rearing entire males and avoiding welfare problems at the same time (Lundström and Zamaratskaia 2006). Therefore, combination of alternatives may be necessary, including genetic selection, improvement of management practices and nutrition, and reduction of age at slaughter (before sexual maturity) (Valeeva et al. 2009). Establishment of suitable thresholds for boar taint compounds, elaboration of precise and fast detection methods for sorting out boar tainted carcass, and refinement or identification of reliable strategies for control of boar taint are needed in future research. Besides, animal health and welfare, meat quality, food safety, and economic feasibility should be taken into account when implementation any alternative.
2.5. Dietary fiber to reduce skatole
High skatole concentrations in adipose tissue is a result of : 1) high amount of TRP (either from diet or cell debris) available in the hindgut for skatole formation, 2) high number or activity of skatole-producing bacteria in the hidngut, 3) insufficient carbohydrate for energy, leading to increased protein fermentation with more TRP degraded to skatole instead of being used in the synthesis of microbial biomass, 4) high absorption rate from the hindgut to portal blood, 5) reduced skatole degradation in liver, and 6) low skatole turnover in adipose tissue (Wesoly and Weiler 2012). Addition of certain highly fermentable DF to the feed reduces skatole production.
2.5.1. Dietary fiber
The definition of dietary fiber (DF) is continuously debated. The CODEX (2009) defines DF as carbohydrate polymers with ten or more monomeric units, which are not hydrolyzed by the endogenous enzymes in the small intestine of humans. European Food Safety Authority (EFSA) (2009) defines DF as non-digestible carbohydrates plus lignin, including all carbohydrate components occurring in foods that are non-digestible in the human small intestine and pass into the large intestine (Jones 2014). Non-digestible carbohydrates consist of three main categories of carbohydrates: 1) resistant oligosaccharides, 2) resistant starch (RS), and 3) non-starch polysaccharides (NSP) (Table 1) (Sajilata et al. 2006; Bindelle et al. 2008; Metzler and Mosenthin 2008).
12 Table 1 Classification of common non-digestible carbohydrates.
Type of carbohydrate Fermentability Common sources in pig diets
Oligosaccharides (2 < DP < 10)
Fructo- and galacto-oligosaccharides High Soybean meal, peas, rapeseed meal,
cereals, milk products Polysaccharides DP > 10
Resistant starch (RS)
Physical inaccessible starch (RS1) Low Grains, seeds, legumes
Crystalline resistant granules (RS2) Low Raw potato, sweet potato, plantain, maize
Retrograded amylose (RS3) Low Cooled heat-treated starchy products
Chemically modified starch (RS4) Low Breads, cakes
Non-starch polysaccharides (NSP)
Cellulose Low Cereals, legumes, forages, plant cell wall Hemicellulose Low Cereals, legumes hulls
Pectins High Fruits, chicory, sugar beet pulp
β-Glucans High Barley, oats, rye
Inulins and fructans High Chicory, Jerusalem artichoke, yam, rye
DP: degree of polymerization.
Consumption of prebiotic inulin-type fructans (ITF; oligofructose and inulin) has been given attention due to its beneficial health impacts in humans and animals. Inulin is widely distributed in a variety of plants, among which are the tubers of Cichorium intybus (chicory roots) and Helianthus tuberosus (Jerusalem artichoke) (Apolinário et al. 2014). Chicory roots contain approximately 150 - 200 g/kg inulin (average DP = 12, range from 2 to 60) and 80 - 120 g/kg oligofructose (average DP = 4, range from 2 - 8) (Flickinger et al. 2003; Roberfroid 2005). ITF are mostly or exclusively β-(2→1) fructosyl-fructose linkages, which are resistant to hydrolysis by enzymes in the small intestine, but can easily be degraded by β-fructofuranosidases produced by certain types of bacteria such as
13 Bifidobacterium species, and Roseburia inulinivorans, etc in the hindgut (Roberfroid 2007; Rivière et al. 2016). Beneficial effects of ITF include increased stool frequency, satiety hormones secretion and mineral (Ca/Mg) absorption, decreased proteolytic activity and ammonia emission, and a beneficial modification of gut microbiota and immune response (Schaafsma and Slavin 2015; Kozłowska et al. 2016; Micka et al. 2016). The bifidogenic and butyrogenic effects of ITF have been well established (De Vuyst and Leroy 2011; Rivière et al. 2016).
2.5.2. Effects of dietary fiber on skatole production
In general, as reviewed by reviewed by (Wesoly and Weiler 2012; Bilić-Šobot et al. 2014; Jensen et al. 2014), the addition of different types of DF (e.g fructo-oligosaccharides (FOS), inulin, pectin and raw potato starch (RPS), and various feed ingredients rich in DF e.g. (chicory roots, jerusalem artichokes, sugar beet pulp, and lupines) to growing pig diets has been shown to reduce skatole formation in the large intestine, the absorption of skatole to the portal vein, and the deposition in adipose tissue (Table 2).
In summary, the most promising DF for reducing skatole levels in entire male pigs seems to be purified inulin or inulin rich feed components such as chicory root or jerusalem artichoke (Jensen et al. 2014). Adding 15-25% chicory to pig feed minimum 14 days before slaughter is thus recommended to reduce boar taint (Hansen et al. 2006; Byrne et al. 2008). Supplementation with RPS has also proved to be effective in reducing skatole levels, if at least 20% is added to the diet. However, reducing its amount to 10% of the diet resulted in no significant skatole reduction (Wesoly and Weiler 2012). Moreover, pelleted RPS has no effect on skatole production most likely because the high temperature during the pelleting process gelatinizes the starch, resulting in increased ileal digestibility (Øverland et al. 2011). Sugar beet pulp, lupine, and FOS have also the potential to reduce skatole in entire male pigs. However, further investigations are needed in order to establish the optimal dosage and the duration of feeding.
14 Table 2 Effect of dietary fiber on skatole in digesta, feces, plasma and adipose tissue.
Feed components % in diet Duration (d) Reduction of skatole Reference Digesta/ Plasma Adipose
feces tissue Inulin 3.9 14 + n.d. n.d. (Rideout et al. 2004) 16 42 n.d. + + (Hansen et al. 2006)
14 42 n.d. + + (Byrne et al. 2008) 3.3 28-42 n.d. n.d. - (Aluwé et al. 2009) ≥ 4.2 28 + n.d. + (Kjos et al. 2010) 6.3 30 + n.d. - (Øverland et al. 2011) 6.8 7 - n.d. - (Vhile et al. 2012) Chicory root ≥ 2.8 ≥7 n.d. + + (Hansen et al. 2006) 10 7-14 n.d. n.d. - (Nielsen et al. 2007) 10-13.3 7-14 n.d. - - (Hansen et al. 2008) 25 28-63 n.d. + + (Byrne et al. 2008) 10 16 n.d. - - (Rasmussen et al. 2012) 9 14 n.d. n.d. + (Zammerini et al. 2012) 5 10 n.d. n.d. + (Aluwé et al. 2013) 15 14 n.d. n.d. + (Maribo 2013) Jerusalem artichoke 12.2 7 + n.d. - (Vhile et al. 2012)
Raw potato starch 10 14 n.d. + n.d. (Jensen and Jensen 1998) artichoke 58 19 + + + (Claus et al. 2003) 60 14 n.d. n.d. + (Kristina Andersson et al. 2005) 60 14 n.d. + + (Zamaratskaia et al. 2005a) ≥ 30 14-21 - n.d + (Lösel and Claus 2005) 30 / + + + (Lösel et al. 2006) 60 14 n.d. + + (Chen et al. 2007) 30 7 n.d. - + (Pauly et al. 2008)
15
15 10 28-42 n.d. n.d. - (Aluwé et al. 2009) 30 7 n.d. n.d. + (Pauly et al. 2010) 20 14 + n.d. + (Øverland et al. 2011) 23, 28 100 + n.d. n.d. (Zhou et al. 2016) Sugar beet pulp 40 17 - n.d. - (Hawe et al. 1992) 20 / + n.d. + (Jensen et al. 1995a) 10 14 n.d. - n.d. (Jensen and Jensen 1998) 9 21-35 n.d. n.d. + (Kjeldsen and Udesen 1998) 15 / n.d. n.d. - (Oeckel et al. 1998) 10 30 + + + (Knarreborg et al. 2002) 20 30 n.d. + + (Whittington et al. 2004) Lupine 10 14 n.d. + n.d. (Jensen and Jensen 1998) 25 7-14 n.d. n.d. + (Nielsen et al. 2007) 10 28-42 n.d. n.d. - (Aluwé et al. 2009) 25 7/14 n.d. + + (Hansen et al. 2008) 15 14 n.d. n.d. - (Maribo 2013) Fructo-oligosaccharide 10 14 n.d. + n.d. (Jensen and Jensen 1998)
0.2 42 - n.d. + (Salmon and Edwards 2015)
/ : not available; +: skatole concentration decreased (p < 0.05); -: no effect (p > 0.05) ; n.d.: not defined
16
16 2.5.3. Mechanisms for the reducing effect of dietary fiber on skatole production
Skatole formation in the pig hindgut is the result of a high amount of available TRP, insufficient available carbohydrates for energy production, and increased number or activity of skatole-producing bacteria. The exact mechanism behind the reducing effect of DF feeding on skatole production in the hindgut has not been established, but several hypotheses have been put forward:
• The butyrate formation theory.
Tryptophan, the precursor of skatole, originates mainly from gut-mucosa cell debris and not from undigested dietary proteins (Claus et al. 1994; Claus et al. 1996). Inulin-type fructans or RPS decrease skatole production due to an increased butyrate production (butyrogenic effect) (Falony et al. 2009; Rivière et al. 2016) that reduces enterocyte apoptosis, resulting in less endogenous TRP from cell debris being available for skatole production by microbes in the pig colon (Claus et al. 2003).
• The microbial activity theory.
The presence of sufficient DF in the hindgut increases the microbial activity, resulting in more TRP being incorporated into microbial biomass (Jensen et al. 1995a; Xu et al. 2002). Further, increased amounts of carbohydrates will decrease the activity of proteolytic bacteria resulting in less TRP available for skatole production (Jensen 2006).
• The microbial population theory.
Adding high levels of DF to the feed reduces the growth of the skatole-producing bacteria in the hindgut of pigs, and hence reduces skatole synthesis (Vhile et al. 2012).
17
3. Hypothesis and objectives
Boar taint, caused primarily by accumulation of skatole and androstenone in fat, is an off-odor and off-flavor released upon heating of meat from uncastrated sexually mature male pigs. Skatole, exhibiting a fecal-like odor, is produced by anaerobic microbial degradation of TRP via decarboxylation of IAA in the hindgut of pigs. Few bacteria have been reported to catalyze the step from IAA to skatole (Deslandes et al. 2001), and no skatole-producing bacterium has been isolated from the pig gut so far. The lack of knowledge on the specialized gastrointestinal skatole-producing bacteria inhabiting the pig hindgut hinders further efforts to control boar taint. Currently, surgical castration of male piglets is a common practice in pig production worldwide to prevent boar taint. However, due to animal welfare concerns, there is an increased desire to stop surgical castration in the European Union by 2018. One strategy to reduce skatole production in entire male pigs is adding fermentable DF to the feed. The link between consumption of DF, so far INF being the most effective, the increase of butyrate production, and the reduction of skatole production in the hindgut of pigs remains unclear.
The following hypotheses were tested in the current project:
1) Skatole production in the hindgut is limited to very few bacterial species. 2) The number of skatole-producing bacteria in the hindgut is affected by DF. 3) The production of skatole is affected by butyrate concentration in the hindgut. 4) The reducing effect of DF on skatole production in the hindgut of pigs is due to more TRP being incorporated into bacterial biomass.
The objectives of this project were to:
1) Screen and characterize bacteria isolated from pig gut able to produce skatole. 2) Report the draft genome sequences of skatole-producing bacteria to provide genetic information for exploring the skatole metabolic pathways 3) Develop a real-time TaqMan-MGB assay for detecting the skatole-producing bacteria in the GI content of pigs. 4) To characterize butyrate-producing bacteria isolated from the pig intestine. Given the possible relation between the concentration of butyrate in the hindgut and the production of skatole. 5) Elucidate mechanisms behind the reducing effect of fermentable DF on skatole production in the hindgut of pigs.
18
4. Methodological consideration
4.1. Standards for describing new taxa of bacteria
In order to describe a new taxon (e.g. Olsenella scatoligenes sp. nov.,), a polyphasic approach, which integrates genomic and phylogenetic data with phenotypic and chemotaxonomic data needs to be applied (paper I). Nomenclature is governed by the Bacteriological Code. Besides, the ‘Rules and Recommendations of the International Code of Nomenclature of Bacteria’ (Lapage et al. 1992) can provide guidance in naming scientific taxa. Type strains should be deposited, before publication, in two different recognized culture collections in different countries. It is of central importance to include all type strains that are relevant to a study. The descriptions of new taxa should preferably be published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). The descriptive characters are summarized in Table 3 according to (Logan et al. 2009; Tindall et al. 2010; Mattarelli et al. 2014).
It is worth noting that novel species descriptions should preferably be based on more than one strain, and so improve the robustness of the species description (Logan et al. 2009). Unfortunately, when classifying O. scatoligenes, we had isolated only one strain (SK9K4), and we were not able to obtain the two undescribed isolates (Olsenella sp. BS-3 and Bacterium OL-1), clustering close together with SK9K4. However, we clearly find strain SK9K4 as being of high biological significance and importance due to its specialized and rather unique metabolism leading to skatole production, which in our opinion, justifies its description and classification as a novel species even though it is based on a single strain.
19
Table 3 Standards for description of new taxa.
Essential Characteristic Additional
Genotypic criteria
16S rRNA gene sequence and similarity value Complementary phylogenetic markers
DNAPhylogenetic G+C content, analysis mol% Nucleic acid fingerprinting
DNA-DNA hybridization Whole genome sequences
Pheno(when typic strains criteria share > 97 % 16S rRNA gene sequence similarity) Cell morphology and arrangement Antibiotic susceptibility
Colony morphology Flagellation type
Spore formation
Motility
Gram-staining reaction
Growth medium and physiological properties (oxidase, catalase) Growth conditions
Biochemical(Temperature, criteria pH, salinity, oxygen)
Enzymatic activities (API tests or Biolog) Indole/skatole production from tryptophan Fermentation pattern of carbohydrates
Fermentation pattern of glucose
Chemotaxonomic criteria
Peptidoglycan MALDI-TOF MS
Fatty acids analysis Polar lipids analysis
Respiratory quinone pattern
Ecological criteria
Source and habitat
Abbreviation: MALDI-TOF MS, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry
20
4.2. Techniques for studying the gut microbiome
The gut microbiota is a complex and dynamic ecosystem that co-evolves with the host from birth and profoundly influences host’s health and disease. The GI-tract of pigs is inhabited by a highly diverse microbiota comprising around 400 species (Leser et al. 2002), with total counts of more than 1010 CFU/g digesta in the hindgut (Butine and Leedle 1989). Until the 1990s, culture was the only technique available to characterize gut microbiota composition and knowledge of the gut microbiota was limited to only the fraction of the gut microbiota that had been cultured (Fraher et al. 2012). To date, over 1000 different microbial species that can reside in the human GI-tract have been identified (Rajilić-Stojanović and de Vos 2014). However, much less is known about pig gut microbiota compared to humans. The development of culture-independent techniques and next-generation high throughput DNA sequencing techniques have spearheaded our knowledge of the complex microbiota such as those found in the pig gut, showing a much higher diversity than described previously by cultivation (Kim et al. 2011; Niu et al. 2015).
Understanding the gut microbiota will continue to benefit from a range of culture-dependent and culture-independent approaches, both of which should ideally be used to complement each other. On one hand, putative functions of genes identified during metagenomic screening can be predicted from comparative analysis to genes of known function and be finally validated with cultured strains. On the other hand, genomic data could provide hints on metabolic needs of the uncultured microbes and aid in improving strategies for the growth of previously uncultivated strains (Walker et al. 2014).
4.2.1. Culture-dependent techniques
Although cultivation of the gastrointestinal microbiota is laborious, it is extremely useful for understanding the detailed physiological and biochemical characterization of the individual isolates. Deployment of novel cultivation approaches allows to expand microbial culture collections and opens up new avenues for microbiome research, and enables direct association of specific phenotypic properties to specific strains (Sommer 2015).
In our project, using 11 different media, a total of 2678 isolates were collected from the pig GI-tract. Based on 16S rRNA gene sequencing, these isolates were categorized into 122 operational taxonomic units (OTUs). The representatives of each 122 OTUs were incubated (37°C, 1 wk) in triplicate in Hungate tubes with PYG-mod medium without or with supplemented IAA, or p-hydroxyphenylacetic. Metabolic end products of these isolates were analyzed by HPLC or GC for screening their ability to produce skatole, p-cresol or butyrate (paper I &V).
21
Culture-dependent methods aiming at determining diversity in the GI-tract present limitations, because they are biased due to selectively allowing growth of some species when their metabolic and physiological requirements can be reproduced in vitro, while suppressing growth of others. Culturing fails to reproduce the ecological niches and symbiotic relationships encountered in the gut required to support the full spectrum of microbial diversity, resulting in underestimated gut microbiota abundance (Carraro et al. 2011). For example, in paper V, Faecalibacterium (F.) prausnitzii only accounted for 0.1% of the total isolates due to this bacterium being notoriously difficult to cultivate and preserve (Li et al. 2016a), whereas data from 16S rRNA gene libraries of pig intestinal microbiota have shown that F. prausnitzii comprised up to 2.9% of the total bacterial population (Leser et al. 2002). F. prausnitzii may thus have been underrepresented in our study.
Notably, the recent high-throughput culturing (culturomic) studies have proven that cultivation can be used as a powerful approach in discovering currently unknown GI microbes (Lagier et al. 2012; Rettedal et al. 2014; Lagier et al. 2015). Lagier et al. (2012) used 212 different cultivation conditions and found that only 51 out of a total of 571 species identified overlapped between culturing and 16S rDNA sequencing methods, highlighting the need for using both culture-dependent and culture-independent approaches to study the gut microbiome. Moreover, having strains in culture facilitates their genome sequencing, thus expanding the reference databases and contributing to the interpretation of physiological and metagenomic data (Walker et al. 2014).
4.2.2. Quantitative real-time PCR
The 16S rRNA gene is a suitable marker gene for taxonomic and phylogenetic quantification of gut microbiota. Examples of the techniques based on 16S rRNA amplicon are quantitative real-time PCR (qPCR), denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), fluorescence in situ hybridization (FISH), DNA microarrays, and high-throughput sequencing (HTS) technologies (Fraher et al. 2012). One shortcoming of current HTS methods is the limitation in their ability to resolve bacteria at or below the genus level due to short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA gene (Ruegger et al. 2014). Besides, techniques like T-RFLP, DGGE/TGGE, FISH, an HTS, which are semi-quantitative, are useful when aiming at studying microbial profiles (Ott et al. 2004). Whereas for aiming at quantifying specific species or groups of bacteria, qPCR with species-specific primers and probes is a more accurate and sensitive method.
Therefore, in this project (paper IV), we developed a TaqMan minor groove binder (MGB) qPCR assay targeting the 16S rRNA gene of O. scatoligenes SK9K4T to quantify this species in the pig
22 gut. SYBR Green qPCR was not adopted because primer pair alone was not sufficient to discriminate O. scatoligenes from O. profusa or O. uli due to high sequence homology in 16s rRNA genes. Taqman-MGB qPCR was thus preferred, since MGB-probe could increase specificity and sensitivity of qPCR assay by allowing for a single-base mismatch discrimination (Van Hoeyveld et al. 2004; Mingxiao et al. 2013).
Efficiency and accuracy of the qPCR depends on DNA quality. Two main obstacles for good DNA quality are inefficient recovery of total gDNA from the bacterial community and presence of PCR inhibitory compounds from the environmental matrix (Zoetendal et al. 2001). Incomplete lysis of the bacteria in a sample or the presence of inhibitors reduce the number of genomic copies available for PCR or reduce the efficiency of amplification (Coyne et al. 2005). Low recovery of DNA is common and seems difficult to avoid, which was demonstrated by a study using different DNA extraction methods for analysis of cecal microbiota, resulting in 64-99.9% loss of the retrievable DNA (Scupham et al. 2007). The low gDNA recovery ( 5%-10%) in our study (paper IV) may partly be caused by long-term (~ 2 years) storage of digesta samples at -20 ˚C.
Optimizing sample storage (e.g. stored completely desiccated) and DNA extraction methods (e.g. including a step for the mechanical disruption of microbial cells by bead beating) (Yuan et al. 2012) are demanded in future studies. Commercial QIAamp® DNA Stool kit, as used in our study, has shown high extraction efficiency and PCR-compatibility, and is frequently used in the extraction of gDNA from human feces (McOrist et al. 2002; Nechvatal et al. 2008; Salonen et al. 2010). However, QIAamp® kit uses a chemical lysis procedure that may lead to incomplete cell lysis. Furthermore, after comparing several DNA extraction methods, FastDNA(™) SPIN Kit for Soil (MP Biomedical), instead of QIAamp® DNA Stool kit was recommended by Burbach et al. (2016) as a suitable DNA extraction kit for the analyses of porcine gastrointestinal tract samples.
4.2.3. Next-generation sequencing
Next-generation sequencing (NGS) is a term used for non-Sanger-based sequencing technology that enables HTS of millions/billions of DNA fragments without a need for cloning (Schuster 2008). Due to the advancing NGS technologies and the falling cost, gut microbiota research has been revolutionized over the past decades (Frank and Pace 2008). Among the NGS technologies, Genome Analyzer/HiSeq 2000/MiSeq (Illumina), 454 GS FLX Titanium/GS Junior (Roche), and SOLiD/Ion Torrent PGM (Life Sciences), are the most representative (Liu et al. 2012b). There are four microbial sequencing methods available so far, namely, whole-genome shotgun (WGS) microbial sequencing, marker gene metagenomics (e.g.16S rRNA gene sequencing), metagenomics,
23 and transcriptomics (RNA-seq). 16S rRNA gene sequencing and metagenomic sequencing are the two main NGS strategies used to explore gut microbiota (Zhang et al. 2016).
In this project, WGS strategy was used to sequence genomic DNA from O. scatoligenes SK9K4T (Illumina HiSeq 2000 platform, Beijing Genomics Institute [BGI], Shenzhen, China) (paper II), and Megasphaera sp. strain DJF_B143 (Illumina MiSeq platform, Center for Geomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmark) (paper III). The latter platform was also used for 16S rRNA gene amplicon sequencing (paper VI).
4.2.3.1. Whole genome sequencing
Rapid advances in sequencing technology and bioinformatic tools during the last decade, has resulted in an exponential increase in draft/complete genome deposits in public databases (Barbosa et al. 2014). Genome sequencing is a stepping stone to carry out a comparative genomic analysis, which is powerful in identifying orthologous genes in species, specific genes, evolutionary signals, and candidate genes associated with pathogenicity, adaptability, and economic significances (Ali et al. 2013). Besides, comparative genomic analysis also applies in designing vaccines, diagnostics and drug development against pathogenic bacteria (Ali et al. 2013).
In our project (paper II & III), both genome sequences were as high-quality draft. As estimated by the software Check M, genomes of O. scatoligenes SK9K4T, and Megasphaera sp. strain DJF_B143 were 99.0% and 99.9% complete, respectively. One of the drawbacks of a draft genome is that there is a risk that the regions of highest interest might not be correctly represented or absent. For example, in our case, if draft genomes were used when conducting a comparative genomic analysis, it might be difficult/impossible to identify candidate genes for skatole production. Thus, as stated by Fraser et al. (2002), comparative genomics is meaningful only in terms of complete genome sequences . In future studies, the painstaking process of completing the O. scatoligenes genome needs to be undertaken.
The relatively short read length produced by most NGS platforms limits their use in genome assembly or finishing (Ye et al. 2015). Currently, long-read sequencing technology such as single-molecule real-time (SMRT) sequencing (Pacific Biosciences), and Oxford nanopore sequencing (the third generation sequencing) are emerging. But it is associated with error rates as high as 15%, rising concerns about its utility (Van Dijk et al. 2014; Goodwin et al. 2016). However, with the optimization of the long-read sequencing technology, it can be an ideal tool to finish genome assemblies or to improve existing draft genomes.
24
4.2.3.2. 16S rRNA gene amplicon sequencing
The 16S rRNA gene amplicon sequencing technique is based on the amplification of small fragments of hypervariable regions of the 16S rRNA gene. The sequences of these fragments are then obtained and compared with reference databases for taxonomic identification (Galan et al. 2016). When performing 16S rRNA gene amplicon sequencing, sample handling, DNA extraction, primer efficiency, PCR amplification, sequencing platform and bioinformatics pipeline can all introduce biases and affect metagenomic analysis (Huse et al. 2012; Galan et al. 2016). Illumina is currently the state of the art when it comes to 16S rRNA gene sequencing. It surpasses the previous 454 technology most notably due to its lower costs, higher accuracy and greater throughput (Sinclair et al. 2015). Currently, the most common approach for studying microbial communities is to use either V4, V3–V4 or V4–V5 primers on Illumina platforms to generate 16S rRNA gene sequences, averaging ~250 - 430"bp in read length (Singer et al. 2016). However, this resolution of sequencing data is unable to reliably provide species level identification. While this limitation affects whole-community analyses to different degrees, it prevents the use of HTS as a tool for identifying and examining individual bacteria of interest.
In this project (paper VI), the V3-V4 regions (recommended based on theoretical analysis (Klindworth et al. 2013)) of the 16S rRNA gene were amplified on the Illumina MiSeq platform, according to the 16S metagenomic sequencing library preparation protocol (Illumina, San Diego, USA). In our study, sequencing data allowed us to compare the relative abundance of the Olsenella genus, but not specifically of the O. scatoligenes species. Therefore, we developed a qPCR assay for quantifying this species. Besides, it is also possible that the reference database (e.g. Greengenes references) is out of date, resulting in some sequences in our 16S datasets being unclassified. Besides, unlike metagenomics, 16S rRNA gene amplicon sequencing fails to provide direct evidence of a community's functional capabilities. However, recently, approaches like phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) (Langille et al. 2013) or R package Tax4Fun (Aßhauer et al. 2015) are proposed to predict functional profiles of microbial communities using 16S rRNA gene sequencing data.
4.2.3.3. Bioinformatic analysis
The wealth of data generated by HTS technology requires innovative bioinformatic solutions (Pop and Salzberg 2008). The complete WGS data analysis process is complex, may include quality control checks and pre-processing measures, de novo or reference-based assembly, automated annotation with or without manual improvement, and improving genome quality by gap filling or
25 other such methods (Escobar-Zepeda et al. 2015; Liao et al. 2015). Assembly is a computational process in which short read sequences are compiled into whole genome sequences, and gene annotation is a computational process in which regions of the DNA containing coding sequences (CDSs) are identified and their functions assigned (Kisand and Lettieri 2013). A variety of software is currently available for accomplishing genome assembly and annotation. Choosing appropriate tools for specific parts of the analysis workflow is a non-trivial task, especially for inexperienced users. The genome assembly (e.g. SPAdes (Bankevich et al. 2012)), and annotation softwares (e.g. Prokka (Seemann 2014)) used for WGS analysis in our project are listed in Figure 4.
Figure 4 The pipeline for analyzing whole genome sequencing.
A wide variety of bioinformatics analysis pipelines exist (e.g. Quantitative Insights Into Microbial Ecology (QIIME), mothur, the Ribosomal Database Project Pipeline (RDPipeline), and SILVAngs) for analyzing 16s rRNA gene sequencing data (Oulas et al. 2015; Plummer and Twin 2015). Among them, QIIME (Caporaso et al. 2010) is one of the most frequently used free bioinformatics software
26 for self-contained microbial community analysis and seems to be established as the “gold standard” (Nilakanta et al. 2014) for marker gene metagenomics. The QIIME workflow for conducting pig fecal microbiota analysis in our study is outlined in Figure 5 (Navas-Molina et al. 2013). R package ampvis was also applied to expand upon these analyses (Albertsen et al. 2015).
Figure 5 Overview of Qiime workflow for analyzing marker gene metagenomics.
4.3. In vivo experimental design
The in vivo experiment followed a Latin square design with 3 treatments, 3 periods, and 6 animals, resulting in 2 animals per treatment and period (Figure 6). Cecal catheterization of pigs allowed us to infuse butyrate directly into the hindgut, and to evaluate the effects of chicory root and butyrate on skatole production and gut microbiota separately.
27
Figure 6 Set up of the in vivo study with infusion of butyrate.
The advantages of the in vivo design are as followings: 1) the Latin square design is more economical and animal friendly in that fewer pigs are used; 2) repeated use of the animals has the advantage of comparing treatments within pigs; and, the time effect can be accounted for in the analysis. A Latin square design gives the opportunity to minimize the high variances between individual animals, resulting in a smaller mean square for error.
The drawbacks of the in vivo design are as followings: 1) the Latin square design prolongs the experiment period from two weeks to five weeks due to rotation of treatments; 2) this design allows collection of hindgut samples at slaughter from a very small number of animals (two in this case). Consequently, only fecal samples, but not samples from the hindgut, which may reflect the gut environment more appropriately, were collected.
28
5. Paper I
Olsenella scatoligenes sp. nov., a 3-methylindole- (skatole) and 4-methylphenol- (p-cresol) producing bacterium isolated from pig faeces
Xiaoqiong Li, Rikke Lassen Jensen, Ole Højberg, Nuria Canibe, Bent Borg Jensen
Published in International Journal of Systematic and Evolutionary Microbiology, 2015, 65:1227–33, DOI 10.1099/ijs.0.000083.
29
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The TRP, MP, joining; likelihood; neighbour maximum NJ, laser-desorption/ionization parsimony; ML, imum matrix-assisted spectrometry; MS, mass time-of-flight MALDI-TOF acid; tic [email protected] Li Xiaoqiong Correspondence 08.Saoei h ancnrbtn opudof compound contributing main the is Skatole 2008). , G 05IUMS 2015 A,idl--ctcacid; indole-3-acetic IAA, T sJX905358. is tal. et rne nGetBritain Great in Printed 04.Crety h surgical the Currently, 2014). , p ceo 4mtypeo)are (4-methylphenol) -cresol rpsdadtetp tani SK9K4 is strain name type The the genus. and this proposed within species novel a represents isolate AD-O assetao eletat n h hsooia n iceia characteristics biochemical and physiological the SK9K4 and strain extracts differentiated cell of spectra mass MALDI-TOF RAgn eune,rvae htteioaerpeetdanwlnaewti h genus the within lineage new a represented isolate produced the also Olsenella that it revealed taint; sequences, boar gene to rRNA contributor main the is ( 4-methylphenol which (skatole), 3-methylindole to acid acaoyi ocbclu,wsioae rmpgfee.SK9K4 faeces. pig from isolated was coccobacillus, saccharolytic ftetreseiso h genus 13989 the of species three the of 9. ) N–N eaens auso tanSK9K4 strain of values relatedness DNA–DNA %). 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Table others and Li X. 1230 were tubes The strains. bacterial cultures 37 four at h-old days 72 three the for with incubated of with inoculated ml) tubes were or (0.1 each) without Hungate mM or (0.29 medium analysis, IAA, PYG-mod product supplemented ml 10 end containing metabolic For laiepopaae aieayaiae ecn rlmds,csieayaiae cdpopaaeand phosphatase acid arylamidase, cystine arylamidase, leucine arylamidase, valine phosphatase, alkaline aafo Go from Data nRpdI 2A rflsrpre yKraatz by reported profiles A, 32 ID Rapid In P Y results ZYM API Characteristic cdfcto of Acidification ai D3 results Profile A 32 ID Rapid ao ellrftyacids fatty cellular Major G DNA eclnhdoyi ( hydrolysis Aesculin Profile results A 20 API riiedihydrolase Arginine Naphthol-AS-BI-phosphohydrolase (C8) lipase Esterase (C4) Esterase Salicin Lactose Trehalose Rhamnose Cellobiose b b b a b b ltmlguai cdarylamidase acid glutamic Glutamyl arylamidase acid Pyroglutamic arylamidase Proline decarboxylase acid Glutamic -Glucosidase -Glucosidase -Galactosidase -Glucosidase -Galactosidase-6-phosphate -Galactosidase + rflsrpre yDewhirst by reported Profiles . ifrnilcaatrsiso tanSK9K4 strain of characteristics Differential otn (mol%) content C ¨ b ker -glucuronidase, T 2, ; tal. et .uli O. tal. et b -glucosidase) (2010). (2001). S 7084 DSM p HA,o A and IAA or -HPAA, N a -acetyl- -chymotrypsin, u n ape eetknat taken were samples and C T 3, ; b tal. et -glucosaminidase, tal. et .profusa O. 20)wr 02375for 2012033705 were (2001) 21)wr 0602for 40060002 were (2011) tal. et a -galactosidase, S 13989 DSM 21)wr 02775for 2012073705 were (2011) C T 4:0; : 14 767772016473705 0716477717 p 64024(/)40*4360*44104022* 46346002* 44(1/3)44002* 46346012 n h yesriso lsl eae pce ftegenus the of species related closely of strains type the and -HPAA a fcsds,terdcino irt n noepouto n oiiefrmannose for positive and production indole and nitrate of reduction the -fucosidase, 62.1 C + + + + +++ + + +++ + + + ++ + +++ + + + 2 222 22 1 8:1 : 18 N T ;4 -actetyl- v 33 9 .umbonata O. c .uli O. nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International .uli O. cdadpeoi-adidlccmon nlsswere at analysis organic stored compound for indolic and ml) and taken (1.0 phenolic- analysed Samples and were Data acid software. gas. PEAK359 carrier GC82-22 the ML using as a He using with chromatography (Mikrolab) gas by determined yig,adteH the and syringe, ocnrto fpeoi-adidlccmonswas compounds indolic and phenolic- Canibe of by outlined concentration as GC by determined b -glucosaminidase, 7762for 47776632 , n 56575for 4516053705 and C S 22620 DSM 8:1 : 18 64.7 .uli O. + 22 2 222 2 222 222 22 222 222 222 22 V VVV 2 v d 9 c 73775for 2713473705 , D .profusa O. T a nAI2A l tan eengtv o indole for negative were strains all 20A, API In . mnoiaeand -mannosidase 2 C 2 .profusa O. 4:0; : 14 0016453705 a 20 ocnrto a immediately was concentration guoiae au agn rm0–5 from ranging value A -glucosidase. n 420202for 441240(2/0)2 and u 64§ C + ++ + + .profusa O. ;ai ocnrtoswere concentrations acid C; 2 2 3 8:1 : 18 . v a D 9 fcsds n oiiefor positive and -fucosidase + c a oiie ,negative; –, Positive; , n 42775for 2402073705 and C tal. et 4:0; : 14 2406477705 a Olsenella -galactosidase, .umbonata O. 63–64§ C 20) The (2007). + + + + + + 2 2 2 2 2 2 4 8:1 : 18 v D 9 c a . 65 O. a V - , Downloaded from www.microbiologyresearch.org by IP: 192.38.33.17 On: Thu, 08 Sep 2016 12:38:28 uli umdfaue4cmrssC comprises 4 feature summed at cd mutn o1 rmr ftettlftyaisaein are acids fatty total acids. the fatty of total more or the % of bold. 10 percentage to amounting a acids as Fatty presented are Results study. yesriso lsl eae pce ftegenus the of species related closely of strains type C 098 umdfaue3cmrssC comprises 3 feature summed 10.928; ECL http://ijs.sgmjournals.org ( and acid H acid formic produced acetic of of amounts amounts for minor except acid, to formic organic and major only product primarily the bacteria as acid acid All lactic 3. to Table glucose the in metabolized of shown HPAA are or bacteria IAA tested glucose, Knarreborg from products by end Metabolic described as HPLC (2002). by analysed SK9K4 1, Strains: 2. Table ol o esprtdb h irba dniiainSystem. Identification Microbial the by that C acids comprises separated 1 fatty feature three Summed be or not two of could groups represent features *Summed etr opie C comprises 2 feature o l tan r o shown. not are strains all for % 1 .umbonata O. hsooial,orioaesest evr iia to similar very be to seems and Lactobacillus isolate skatole our produce physiologically, to and able IAA from was rumen & 11201 the Yokoyama by that strain study reported previous (1981) A containing Carlson shown). medium of not PYG-mod any (data in by TRP detected production was skatole strains no the but medium, PYG-mod auae acids Saturated acid Fatty umdfeatures* Summed acids Unsaturated nnw 4991.4 14.959 Unknown eaoie A osaoeand skatole to IAA metabolized 1 1 : C18 2 1 1 : C18 4 3 iso 0 : C19 0 : C18 0 : C16 anteiso 0 : C15 iso 0 : C14 0 : C14 1 0 : C12 1 s I iso 1 : C19 1 6.6 AT12-13 1 : C13 TR rc mut ( amounts Trace , ellrftyai otnso tanSK9K4 strain of contents acid fatty Cellular v v 7 9 c c S 22620 DSM 2 p tan121 u oorkoldethis knowledge our to but 11201, strain sp. rmguoefretto.SK9K4 fermentation. glucose from T p 2, ; HA,rsetvl.Mrhlgclyand Morphologically respectively. -HPAA, .uli O. 4:0 : 14 , 576. 6020.7 16.0 69.8 25.7 25.9 20.7 T O n/rC and/or 3OH ND ND ND ND ND TR TR TR 234 12 2.2 . 5.5 2.5 7.7 1.0 6.2 1.1 4.3 2.7 . . . 1.5 1.6 7.7 3.1 %); 1 S 7084 DSM l aawr bandi h present the in obtained were data All . .uli O. 3:0 : 13 7:1 : 17 , ND hc nypoue trace produced only which , O n/rC and/or 3OH . M.Only mM). 1.0 s n/rC and/or I iso o eetd auso esthan less of Values detected. not , T ND ND ND TR 1.3 1.9 1.6 2.8 1.3 3, ; 6:1 : 16 p .profusa O. HA to -HPAA 6:1 : 16 s n/runknown and/or I iso v 7 5:1 : 15 Lactobacillus 1911.3 11.9 18.4 8720.9 18.7 31.6 18.8 c 7:1 : 17 DND ND RTR TR RTR TR TR TR TR TR TR TR . 2.8 4.5 2.0 2.5 2.6 n/rC and/or S 13989 DSM s ;summed H; iso nes B. anteiso .profusa O. p Olsenella ceo in -cresol T T p n the and and 6:1 : 16 -cresol ND ND tal. et v T sp. 4, ; O. 6c; 34 ntxue raywie pqewt semi-translucent with granular are opaque colonies creamy-white, days, six texture, for in agar Gram-stain- PYG-mod strictly on incuba- are anaerobic tion non-sporulating, After Cells coccobacillus-shaped. strain. and positive single non-motile, a on anaerobic, based is Description adj. part. N.L. producing; ktl;NL uf - suff. N.L. skatole; leel scatoligenes Olsenella eune 9. –36%,dfeetainb MALDI- by gene rRNA differentiation 16S in %), %–93.6 values (92.7 similarity low sequences the summary, In skatole of production that and by taint boar to contribute may SK9K4 Strains:1, 3. Table eaoi n rdc atrscerydmntaethat demonstrate and clearly SK9K4 patterns characteristics strain product physiological, chemotaxonomic in end differences metabolic and as well as biochemical spectra, mass TOF .Temjrclua at cdo el rw nPYG-mod in grown C cells of is acid medium fatty Table cellular in major of Detailed given The S3). are 1. diameters characteristics (Fig. enzymic elevations have and raised biochemical opaque with days, and mm creamy-white, six mm–1.0 margins 0.5 texture, for semi-translucent in agar with granular PYG-mod are on colonies incubation anaerobic Olsen of al. results et the to addition In of description Emended genus the name within species novel ecito of Description of minor amounts TRP, with from trace not acid produces but produces IAA, and from organic and skatole Produces major acid, acid. formic only acetic of the amounts as acid lactic .umbonata O. rdcs oe aeltesidct io rdcs rdcsin Products products. ( minor products indicate trace major indicate indicate letters parentheses letters case Capital acid. lower formic f, products, acid; acetic a, acid; lactic h yesriso lsl eae pce ftegenus the of species related closely of strains Olsenella type the ol eseuae htpouto fsaoeb SK9K4 by skatole it of study, production present that the speculated a of be in results could deposited the or From characterized collection. further culture been not has strain rai cdpouto ,a ,a f ,a ,a f a, L, f a, L, (f) a, L, f a, L, p IAA from production Skatole production acid Organic H Characteristic Ceo rdcinfo HPAA from production -Cresol .uli O. 2 production 20)adKraatz and (2001) leel scatoligenes Olsenella eaoi n rdc rflso tanSK9K4 strain of profiles product end Metabolic a otiuet aioi nhumans. in halitosis to contribute may S 22620 DSM T T 8:1 : 18 p 2, ; ceo from -cresol eisrcgiina h yesri fa of strain type the as recognition merits leel scatoligenes Olsenella v .uli O. genes 9 c T [ Tbe2.Mtblzsguoeto glucose Metabolizes 2). (Table S 7084 DSM . sca.to.li fo r v. Gr. (from , + tal. et scatoligenes oiieo present; or Positive , p o. sproposed. is nov., sp. p leel uli Olsenella -HPAA. 21) efudta after that found we (2011), 9 , ++ ++ 22 ens ..n. N.L. ge.nes. T 23412 leel scatoligenes Olsenella 3, ; . mM). 1.0 Olsenella tal. et .profusa O. skatole-producing gennaio 19) Dewhirst (1991), o hc the which for , ˆ S 13989 DSM p nov. sp. 2 oproduce) to + 22 22 eaie L, negative; scatolum p nov. sp. 2 T 1231 and T 4, ; ] . T , Downloaded from www.microbiologyresearch.org by IP: 192.38.33.17 On: Thu, 08 Sep 2016 12:38:28 h ao ellrftyais( C acids are fatty cellular major The h yesri,SK9K4 strain, type The cdayaiae riiedihydrolase, arginine glutamic arylamidase, glutamyl pyr- acid and proline-, acid- pyroglutamic serine-, phosphatase, tyro- acid-, not histidine-, oglutamic alkaline leucine-, glycine-, is alanine-, phenylalanine-, for nitrate sine-, glycine-, and detected leucyl formed, is arginine-, not not Activity is is reduced. indole acetoin mannose raffinose, from and produced is detected and acid ID32A, not Rapid gelatin is In produced. melezitose, not Urease but hydrolysed. glycerol, Aesculin, is trehalose. arabinose, and sorbitol xylose, raffinose, mannitol, not maltose, but sucrose, rhamnose, from glucose, and cellobiose, from salicin, formed lactose, is mannose, acid 20A, API In u o rmTP n produces and TRP, from not but a sltdfo h acso isa ahsUniversity, G Aarhus at DNA pigs genomic of The faeces Denmark. the from isolated was hiso el r pt 5–6 to up are cells of chains and dase, ehia sitneadMre ole o epi rprn the preparing in help for Poulsen trees. Morten skilful phylogenetic for and Rebsdorf Mona assistance Thomas thank and technical to Poulsen Agency, want Trine AgriFish We Durup, Karin Fisheries. Danish Dinsen, and the programme, Agriculture by Food, RDD funded of Organic is Ministry Organic in It production Research ICROFS. the for pig Systems, Centre of Food ‘Organic International part by project coordinated is is the which that under castration’ carried without was work The Acknowledgements 1–2 are cells raised Single pairs chains. with in singly, short mm i.e. in mm–1.0 arranged, or 0.5 variously are of Cells elevations. diameters and margins others and Li X. 1232 ( I. Kleinberg, on & grain D. fermented Codipilly, ( and B. feed B. piglets. in 85 Jensen, liquid performance growth & fermented and H. ecology gastrointestinal feeding J. of Badsberg, O., Effect Højberg, N., Canibe, References C) naphthol-AS-BI-phosphohydrolase, (C8), N galactosidase-6-phosphate, ini atcai,wt io mut faei cdand acid acetic of amounts H minor no fermenta- with acid; formic glucose acid, of lactic product is end tion major The detected. not uigmldrfraini h aiaysdmn oe ytmand involved. bacteria system oral model the sediment of examination salivary initial the in formation malodor during ltmcai eabxls.N ciiyi eetdfor detected is activity No urease, decarboxylase. acid glutamic iae cdphosphatase, aryla- acid cystine arylamidase, midase, leucine phospha- arylamidase, alkaline valine system, tase, ZYM API the Using dihydrolase. chymotrypsin, tani 21mol%. 62.1 is strain -acetyl- 2959–2971. , 4:0 : 14 b a gcsds r eetd u iae trypsin, lipase, but detected, are -gucosidase -mannosidase, a b -galactosidase, n C and -glucosaminidase, 8:1 : 18 a 2 -galactosidase, spoue.Poue ktl rmIAA, from skatole Produces produced. is v 9 c a . T fcsds n seae(4 are (C4) esterase and -fucosidase a ( -arabinosidase, 5 2008 a guoiaeand -glucosidase C 19907 JCM a m guoiae seaelipase esterase -glucosidase, a long. m . fcsds n arginine and -fucosidase ). 0%o h oa present) total the of % 10 + eeaino indole/skatole of Generation N p -actetyl- otn ftetype the of content C ceo from -cresol T b rahRes Breath J m 5 -galactosidase, nlnt and length in m b S 28304 DSM -glucuronidase, b b -glucosamini- -galactosidase b -glucosidase p nmSci Anim J 2 -HPAA. 017017. , 2007 T b a ), ). - - 35 nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International leel profusa Olsenella uli Lactobacillus niitco h atra omnt nteiemo rie chickens broiler of ileum ages. the various in at community bacterial the on antibiotic rmctxn erae nohla elrsos oinflammatory to response cell endothelial cytokines. decreases toxin, uremic rates. renaturation from hybridization Biochem DNA of measurement he ue n i euu,adeedddsrpin of descriptions emended uli and Olsenella the jejunum, from pig bacterium and acid rumen lactic sheep anaerobic microaerotolerant a nov., sp. aiu ieiodapproach. likelihood maximum iiu hnefraseii retopology. tree specific a for change minimum ut,R . hn .J,Ael,M hi .( Y. Chai, & M. Adeolu, J., W. ( Chen, authors S., other R. Gupta, & D. Bruce, F., 27C). of J. sequence Cheng, genome H., Complete Tice, T., Rio, Go ( M. W. Fitch, ( J. Felsenstein, De G., ( Grau, Y. Berland, V., & Moal, J. C., Sampol, Guilianelli, R., Vanholder, P., R., Brunet, Smet, C., Cerini, L., Dou, J., Downes, B., ( Coleman, G. N., W. Tzellas, Wade, & J., A. B. D. Spratt, Paster, E., F. Dewhirst, Garie B., ( Deslandes, A. Reynaerts, & H. Cattoir, J., Ley, De raz . alc,R .&Seso,L ( L. Svensson, & J. R. Wallace, M., Kraatz, Tannock, & B. ( B. W. Jensen, M., G. R. Engberg, A., M. Simon, A., Knarreborg, ( M. Kimura, C., S. Park, H., Na, ( M., authors other Kim, & H., H. S. J. Yoon, Lee, K., S., Lee, Y. J., Jeon, Y. Cho, S., O. Kim, C., Bendixen, K., H., M. Callesen, Rasmussen, R., R., ( authors V. Thomsen, other & Gregersen, B., B. Ekstrand, G., A. A. Kudahl, Kongsted, B., B. Jensen, ( B. B. Jensen, & T. M. Jensen, B B. Jensen, & P. R. Cox, T., ( M. H. Jensen, K. Schleifer, & H. Festl, R, A. V. Huß, ae fbs usiuin hog oprtv tde fnucleotide of studies comparative through sequences. substitutions base of rates p 17.Eie yW .Jne.Rsid:Dns etResearch Meat Danish Roskilde: Jensen. K. In W. pigs. Institute. by male Edited entire 41–75. of pp. tract digestive the in family class Atopobiaceae the of Coriobacteriales division for class the for signatures saoe n noepouto ymxdppltoso i fecal pig of populations mixed by production bacteria. indole and (skatole) rkroi 6 RAgn eunedtbs ihphylotypes with database sequence gene species. rRNA uncultured represent that 16S prokaryotic a irbooia n iceia fet fsaoeo animal on skatole of effects biochemical production. and microbiological rlioae n lnd1SrN eune htfl ntefamily the in fall that sequences rDNA 16S Coriobacteriaceae cloned and isolates oral pcrpooercdtriaino N yrdzto from hybridization DNA of rates. renaturation determination spectrophotometric eot42. Report Review Position In ¨ e,M,Hl,B,Lcs . oa,M,Yswn,M,GaiaDel Glavina M., Yasawong, M., Nolan, S., Lucas, B., Held, M., ker, lentvst ugclCsrto nDns i Production Pig Danish in Castration Surgical to Alternatives tn eoi Sci Genomic Stand 2002 Eggerthellaceae 12 plEvrnMicrobiol Environ Appl inyInt Kidney o Evol Mol J 133–142. , ). and ietPo Sci Prod Livest 1980 a.nv,and nov., fam. fet fdeayftsuc n uteaetclvl of levels subtherapeutic and source fat dietary of Effects otiigteeeddfamily emended the containing , p 72.Dns etefrFo n Agriculture and Food for Centre Danish 17–27. pp. , 1971 plEvrnMicrobiol Environ Appl leel profusa Olsenella 1981 ecito of description : p nov. sp. as ). ytAp Microbiol Appl Syst ). leel uli Olsenella a.nov. fam. ipemto o siaigevolutionary estimating for method simple A 62 ). 16 ´ Coriobacteriia y .&Hue .( A. Houde, & C. py, oaddfnn h oreo evolution: of course the defining Toward 1999–2009. , vltoaytesfo N eune:a sequences: DNA from trees Evolutionary 111–120. , 3 n ytEo Microbiol Evol Syst J Int 76–84. , 71 Coriobacteriia 2001 Eggerthellales 1998 193–200. , n ytEo Microbiol Evol Syst J Int 2014 leel uli Olsenella Olsenella . o Evol Mol J 61 n ytEo Microbiol Evol Syst J Int n ytEo Microbiol Evol Syst J Int ). 3180–3184. , ). ob o.addsrpinof description and nov. comb. hrceiaino oe human novel of Characterization ). n t ifrn lds proposal clades; different its and irba rdcino skatole of production Microbial 2012 nitouto oba taint. boar to introduction An 68 4 184–192. , 5918–5924. , e.nv,rcasfcto of reclassification nov., gen. 2011 noteeeddorder emended the into r.nv,cnann the containing nov., ord. . 17 ). 1970 yesri VID76D- (VPI strain type ( ktl n orTaint Boar and Skatole nrdcn EzTaxon-e: Introducing 1995 1983 368–376. , ytZool Syst ). Coriobacteriaceae 2001 ). leel umbonata Olsenella 51 2002 ). ). 2013 h quantitative The 1797–1804. , 3-Methylindole tde nthe on Studies 63 ). 62 20 ). 61 3379–3397. , ). eiwof Review 716–721. , P 406–416. , 795–803. , Molecular Olsenella, ceo,a -cresol, 2010 u J Eur and - 65 A ). , Downloaded from www.microbiologyresearch.org by IP: 192.38.33.17 On: Thu, 08 Sep 2016 12:38:28 efrac iudchromatography. liquid performance G the of measurement fbceilwoecl at cdmty ses nldn hydroxy including esters, methyl acid fatty acids. whole-cell bacterial of ehdfrrcntutn hlgntctrees. 425. phylogenetic reconstructing for method itiuinadfnto fidl--ctcai isnhtcpath- biosynthetic acid indole-3-acetic of function and distribution asi bacteria. in ways http://ijs.sgmjournals.org ( ( M. J. Nei, & T. N. Coulson, Saitou, & J. A. ( Blakney, E. L., W. C. of Moore, Patten, descriptions & emended V. and minutus crevice L. gingival Moore, human L., J. uli Lactobacillus Johnson, I., Olsen, ( J. M. Wolin, & L. T. Miller, from samples ( T. L. gastrointestinal Miller, in ( B. bifidobacteria B. piglets. Jensen, & of M. Jakobsen, Enumeration C., Bendixen, L., L. Mikkelsen, ( B. W. Whitman, & U. Premachandran, M., Mesbah, ( E. T. Devine, & J. J. O’neill, A., M. Roy, D., L. Kuykendall, cd,atboi eitne n exrbncecai homology acid deoxyribonucleic and of resistance, groups antibiotic acids, ugt ehiu o utvtn biaeanaerobes. obligate 27 cultivating for technique Hungate 985–987. , lnMicrobiol Clin J plEvrnMicrobiol Environ Appl and rdrioimjaponicum Bradyrhizobium 1982 tetccu parvulus Streptococcus p o.and nov. sp. rtRvMicrobiol Rev Crit ). igedrvtzto ehdfrruieanalysis routine for method derivatization Single + 16 1987 otn fdoyiouli cdb high- by acid deoxyribonucleic of content C 584–586. , 1974 ). h egbrjiigmto:anew a method: neighbor-joining The atbclu rimae Lactobacillus 69 ). 654–658. , eu otemdfcto fthe of modification bottle serum A . n ytBacteriol Syst J Int 39 . n ytBacteriol Syst J Int n ytBacteriol Syst J Int 395–415. , o ilEvol Biol Mol p o.fo the from nov. sp. 2013 1989 plMicrobiol Appl 41 39 38 1988 lactobacillus 261–266. , ). 159–167. , 358–361. , ). Activity, 4 Precise ). 1991 2003 406– , Fatty ). ). 36 71–76. rumen a by para-cresol o. e ampoebceimioae from isolated 976–981. gammaproteobacterium new magnifica a nov., ooaa .T alo,J .( R. J. Carlson, & T. M. Yokoyama, ( R. J. Carlson, & T. M. ( Yokoyama, A. M. Cotta, manure. & acid swine acetogen indoleacetic L. and the H. skatole of Drake, by production P., the for N. pathway Price, Catabolic ( R., S. T. Whitehead, D., G. Martin, Yost, K., & W. Nichols, G. C., J. Moore, Lamb, S., E., N. Cutler, Makin, M., J. Weems, Ma To ( S. Kumar, & A. Filipski, MEGA D., Peterson, G., Stecher, K., Tamura, ( T. G. Macfarlane, & A. E. Smith, uaei n osbeploaycarcinogen. 67. pulmonary possible a and mutagenic n eae noi opud yrmnlmcoraim nvitro. in microorganisms ruminal Microbiol by Appl compounds indolic related and noi opud yaarbcbcei ntehmnlarge human the in bacteria anaerobic by intestine. compounds indolic Evol ´ ´ h .M,Shmn,P,Broi .K,Ke K., A. Borsodi, P., Schumann, M., E. th, ilgt,K ( K. rialigeti, 30 :mlclreouinr eeisaayi eso 6.0. version analysis genetics evolutionary molecular 6: 2725–2729. , irbEcol Microb Dpea Sarcophagidae). (Diptera: 27 plEvrnMicrobiol Environ Appl 2008 540–548. , lsrdu rki lsrdu scatologenes Clostridium drakei, Clostridium ). 33 olarimnschitiniclastica Wohlfahrtiimonas 180–188. , Lactobacillus 1974 1997 1981 leel scatoligenes Olsenella sp. n ytEo Microbiol Evol Syst J Int 74 ). ). ). ismlto ftryptophan of Dissimilation 1950–1953. , omto fpeoi and phenolic of Formation plEvrnMicrobiol Environ Appl 2009 rdcino ktl and skatole of Production ´ i . Kova Z., ki, ). oio Sci Toxicol -ehlnoeis 3-Methylindole e.nv,sp. nov., gen. Wohlfahrtia ´ s .L & L. A. cs, 112 o Biol Mol p nov. sp. 2008 2013 1233 and , 59– , 41 58 ). ). , , 5.1 Supplementary Materials for Paper I
Olsenella skatologenes sp. nov., a 3-methylindole (skatole) and 4-methylphenol (p-cresol) producing bacterium isolated from pig feces
Xiaoqiong Li1*, Rikke Lassen Jensen2, Ole Højberg1, Nuria Canibe1, Bent Borg Jensen1
1Department of Animal Science, Aarhus University, Tjele, Denmark
2Department of Large Animal Science, University of Copenhagen, København, Denmark
Running title: Olsenella skatologenes sp. nov.,
Corresponding author:
*Xiaoqiong Li
Tel.: +45 87154257 Fax: +45 8715 0201
E-mail: [email protected]
37
Table S1. Differential phenotypic characteristics of strain SK9K4T and the type strains of the closely related Olsenella species O. uli DSM 7084T, O. profusa DSM 13989T, and O. umbonata DSM 22620T.
Bacterium SK9K4T * O. uli* O. profusa† O. umbonata† Growth medium PYG-mod PYG-mod FAA/PYG FAA/PYG Colony and cell
morphology:Diameter (mm) ≤ 1 ≤ 1 ≤2/3 ≤3-4/4 Elevation Raised Raised Pulvinate Umbonate Opacity (margin/centre) Semi-translucent Semi-translucent Opaque/opaque Semi-translucent Color /opaqueCream-white /opaqueCream-white Cream-white /opaqueGreyish white Texture Granular Granular Granular Butyrous Cell arrangement Single, pairs, short Single, pairs, short or Short to very long Single, pairs, short or chains longer chains serpentine chains longer chains Gram stain + +# + + Motility – – # – – Sporulation – – # – – Growth at 37°C + + # + + Oxygen requirement Anaerobic Anaerobic# Anaerobic Anaerobic Isolate source Pig gastrointestinal Human gingival Human oral cavity Pig jejunum
tract crevices# *Data obtained from the present study.
# Data from (Dewhirst et al., 2001).
† Data from (Kraatz et al., 2011)
38
38
Supplementary Fig. S1.1. Maximum Likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between the strain SK9K4T and closely related members of the family Atopobiaceae. Bootstrap values (>50%) based on 1000 replicates are listed as percentages at branching points. Bifidobacterium breve ATCC 15700 was used as outgroup. Bar, 0.02 substitutions per nucleotide position. GenBank accession numbers are given in parentheses.
39
Supplementary Fig. S1.2. Maximum-parsimony tree based on 16S rRNA gene sequences, showing the relationships between the strain SK9K4T and the closely related members of the family Atopobiaceae. Bootstrap values (>50%) based on 1000 replicates are listed as percentages at branching points. Bifidobacterium breve ATCC 15700 was used as outgroup. Bar, 20 substitutions per nucleotide position. GenBank accession numbers are given in parentheses.
40
Supplementary Fig. S2. Light micrographs of strain SK9K4T (A) and type strain Olsenella uli DSM 7084T (B) grown in liquid PYG-mod medium at 37 °C for three days.
41
Supplementary Fig. S3. Colony morphologies of strain SK9K4T (A) and type strain Olsenella uli DSM 7084T (B) after incubation on PYG-mod agar at 37 °C for six days.
42
Supplementary Fig. S4. MSP dendrogram generated by BioTyper software (version 3.1, Bruker Daltonics) supplemented with the spectra of Olsenella umbonata DSM 22620T and O. umbonata DSM 22619 (red branch) from the DSMZ-internal spectra data base showing the similarity of MALDI-TOF mass spectra of cell extracts of strains SK9K4T, O. uli DSM 7084T, O. profusa DSM 13989T and O. umbonata DSM 22620T. The distance level is normalized to a maximum value of 1000.
43
6. Paper II
Draft Genome Sequence of Olsenella scatoligenes SK9K4T, a Producer of 3-Methylindole (Skatole) and 4-Methylphenol (p-Cresol), Isolated from Pig Feces
Xiaoqiong Li, Ole Højberg, Samantha Joan Noel, Nuria Canibe, Bent Borg Jensen
Published in Genome Announc 4(1):e00042-16. doi:10.1128/genomeA.00042-16.
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crossmark
Draft Genome Sequence of Olsenella scatoligenes SK9K4T, a Producer of 3-Methylindole (Skatole) and 4-Methylphenol (p-Cresol), Isolated from Pig Feces
Xiaoqiong Li, Ole Højberg, Samantha Joan Noel, Nuria Canibe, Bent Borg Jensen Department of Animal Science, Aarhus University, Tjele, Denmark
Olsenella scatoligenes SK9K4T is a strictly anaerobic bacterium isolated from pig feces that produces the malodorous com- Downloaded from pounds 3-methylindole (skatole) and 4-methylphenol (p-cresol). Here, we report the 2.47 Mbp draft genome sequence of SK9K4T, exploring pathways for the synthesis of skatole and p-cresol from the amino acids tryptophan and tyrosine, respec- tively.
Received 11 January 2016 Accepted 11 January 2016 Published 25 February 2016 Citation Li X, Højberg O, Noel SJ, Canibe N, Jensen BB. 2016. Draft genome sequence of Olsenella scatoligenes SK9K4T, a producer of 3-methylindole (skatole) and 4- methylphenol (p-cresol), isolated from pig feces. Genome Announc 4(1):e00042-16. doi:10.1128/genomeA.00042-16. http://genomea.asm.org/ Copyright © 2016 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Xiaoqiong Li, [email protected].
he cytotoxic and malodorous compounds 3-methylindole Coriobacteriaceae, CheckM v1.0.3 (11) estimated the genome to be T(skatole) and 4-methylphenol (p-cresol) are produced from 99.0% complete. It has a GϩC content of 62.4%, consistent with the anaerobic degradation of L-tryptophan and L-tyrosine, respec- our previous report (62.1 mol%) (6). As expected, the O. scatoli- tively (1, 2). Malodorants from food production animals are both genes genome contains 4-hydroxyphenylacetate decarboxylase (4- public nuisances and health concerns. Skatole is also the main Hpd) (EC 4.1.1.83) genes, verifying that it possesses the tyrosine compound that causes boar taint, which is an offensive odor and degradation IV (to 4-methylphenol) pathway (12). Through com- on September 5, 2016 by Aarhus Univ flavor present in the meat of some male pigs (3). Only a few cul- parative genomics, we have also found that O. uli possesses 4-Hpd tured microorganisms are reported to produce skatole and, up to genes, which has previously been reported in Clostridia only (13). now, only four (Clostridium scatalogenes [4], C. drakei [5], Moreover, O. scatoligenes also has an aliphatic amidase (EC Olsenella scatoligenes [6], and O. uli [7]) have been fully charac- 3.5.1.4) gene, which is required to produce IAA from tryptophan terized and made available in culture collections. Unlike C. scata- (14). However, O. scatoligenes is not able to synthesize IAA (6). logenes and C. drakei, which produce skatole directly from trypto- Since the enzymology of skatole synthesis has not been character- phan (2), O. scatoligenes and O. uli synthesize skatole by ized so far, very little is known about the genetics of this pathway. decarboxylating the intermediate indole-3-acetic acid (IAA), the Nevertheless, candidate genes for skatole production can poten- proximate precursor of skatole (6). Notably, O. scatoligenes is the tially be identified by comparative genomics. only skatole-producing bacterium isolated from the pig gut. Con- Nucleotide sequence accession numbers. The O. scatoligenes sequently, the genome sequence of this bacterium is of interest as SK9K4T genome sequence has been deposited at DDBJ/EMBL/ it could potentially be used to elucidate the skatole metabolic GenBank under the accession number LOJF00000000. The ver- pathways, hence facilitating the reduction of skatole production sion described in this paper is version LOJF01000000. and, thus, boar taint in pigs. Genomic DNA from O. scatoligenes SK9K4T was extracted and ACKNOWLEDGMENT purified as previously described (6). A DNA library with a read This work was supported by the Danish AgriFish Agency, Ministry of length of 90 bp and insert size of 500 bp was constructed and then Food, Agriculture and Fisheries (project 3405-10-OP-00134). X.L. was sequenced on an Illumina HiSeq 2000 platform (Beijing Genom- supported by a scholarship from China Scholarship Council (CSC). ics Institute [BGI], Shenzhen, China), following the manufactur- FUNDING INFORMATION er’s instructions. Removal of adaptors, low-quality reads, poly-N China Scholarship Council (CSC) provided funding to Xiaoqiong Li. sequences, error paired-end reads, and duplications (8) resulted in 250 Mbp of clean data. Reads were assembled into 16 contigs This work was supported by the Danish AgriFish Agency, Ministry of (Ͼ200 bp) with approximately 101ϫ coverage using SPAdes Food, Agriculture and Fisheries (project number 3405-10-OP- v3.6.1 (9) and the draft genome was then annotated using Prokka 00134). The first author was supported by a scholarship from China v1.1 (10). Scholarship Council (CSC). The draft genome sequence of O. scatoligenes has a total length REFERENCES of 2,469,565 bp, and a N50 length of 594,252 bp, comprising 2,111 1. Elsden SR, Hilton MG, Waller JM. 1976. The end products of the me- protein-coding sequences, 3 rRNAs (including 1 5S, 1 16S, and 1 tabolism of aromatic amino acids by Clostridia.ArchMicrobiol107: 23S), and 48 tRNAs genes. Using the gene marker set for the family 283–288. http://dx.doi.org/10.1007/BF00425340.
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2. Whitehead TR, Price NP, Drake HL, Cotta MA. 2008. Catabolic pathway sequences of DA and F344 rats with different susceptibilities to arthritis, for the production of skatole and indoleacetic acid by the acetogen Clos- autoimmunity, inflammation and cancer. Genetics 194:1017–1028. tridium drakei, Clostridium scatologenes, and swine manure. Appl Environ http://dx.doi.org/10.1534/genetics.113.153049. Microbiol 74:1950–1953. http://dx.doi.org/10.1128/AEM.02458-07. 9. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov 3. Jensen MT, Cox RP, Jensen BB. 1995. 3-methylindole (skatole) and AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, indole production by mixed populations of pig fecal bacteria. Appl Envi- Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. ron Microbiol 61:3180–3184. SPAdes: a new genome assembly algorithm and its applications to single- 4. Song Y, Jeong Y, Shin HS, Cho B-K. 2014. Draft genome sequence of cell sequencing. J Comput Biol 19:455–477. Clostridium scatologenes ATCC 25775, a chemolithoautotrophic aceto- 10. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin- genic bacterium producing 3-methylindole and 4-methylphenol. Genome formatics 30:2068–2069. http://dx.doi.org/10.1093/bioinformatics/ Announc 2(3):. http://dx.doi.org/10.1128/genomeA.00459-14. btu153. 5. Jeong Y, Song Y, Shin HS, Cho B-K. 2014. Draft genome sequence of 11. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. acid-tolerant Clostridium drakei SL1T,apotentialchemicalproducer 2015. CheckM : assessing the quality of microbial genomes recovered from through syngas fermentation. Genome Announc 2(3):e00387-14. http:// isolates, single cells, and metagenomes. Genome Res 25:1043–1055. http:// Downloaded from dx.doi.org/10.1128/genomeA.00387-14. dx.doi.org/10.1101/gr.186072.114. 6. Li X, Jensen RL, Højberg O, Canibe N, Jensen BB. 2015. Olsenella 12. Caspi R, Altman T, Billington R, Dreher K, Foerster H, Fulcher CA, scatoligenes sp. nov., a 3-methylindole- (skatole) and 4-methylphenol- Holland TA, Keseler IM, Kothari A, Kubo A, Krummenacker M, (p-cresol) producing bacterium isolated from pig faeces. Int J Syst Evol Latendresse M, Mueller LA, Ong Q, Paley S, Subhraveti P, Weaver DS, Microbiol 65:1227–1233. http://dx.doi.org/10.1099/ijs.0.000083. Weerasinghe D, Zhang P, Karp PD. 2014. The MetaCyc database of 7. Göker M, Held B, Lucas S, Nolan M, Yasawong M, Glavina Del Rio T, metabolic pathways and enzymes and the BioCyc collection of pathway/ Tice H, Cheng J-F, Bruce D, Detter JC, Tapia R, Han C, Goodwin L, genome databases. Nucleic Acids Res 42:D459–D471. http://dx.doi.org/ Pitluck S, Liolios K, Ivanova N, Mavromatis K, Mikhailova N, Pati A, 10.1093/nar/gkt1103. Chen A, Palaniappan K, Land M, Hauser L, Chang Y-J, Jeffries CD, 13. Selmer T, Andrei PI. 2001. p-hydroxyphenylacetate decarboxylase from
Rohde M, Sikorski J, Pukall R, Woyke T, Bristow J, Eisen JA, Markow- Clostridium difficile. A novel glycyl radical enzyme catalysing the forma- http://genomea.asm.org/ itz V, Hugenholtz P, Kyrpides NC, Klenk H-P, Lapidus A. 2010. Com- tion of p-cresol. Eur J Biochem 268:1363–1372. http://dx.doi.org/10.1046/ plete genome sequence of Olsenella uli type strain (VPI D76D-27C). Stand j.1432-1327.2001.02001.x. Genomic Sci 3:76–84. http://dx.doi.org/10.4056/sigs.1082860. 14. Comai L, Kosuge T. 1980. Involvement of plasmid deoxyribonucleic acid 8. Guo X, Brenner M, Zhang X, Laragione T, Tai S, Li Y, Bu JB, Yin Y, in indoleacetic acid synthesis in Pseudomonas savastanoi. J Bacteriol 143: Shah AA, Kwan K, Li Y, Jun W, Gulko PS. 2013. Whole-genome 950–957. on September 5, 2016 by Aarhus Univ
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46 7. Paper III
Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole
Xiaoqiong Li, Ian P. G. Marshall, Lars Schreiber, Ole Højberg, Nuria Canibe, Bent Borg Jensen
Published in Genome Announc 4(1):e00007-16. doi:10.1128/genomeA.00007-16.
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Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole
Xiaoqiong Li,a Ian P. G. Marshall,b Lars Schreiber,b Ole Højberg,a Nuria Canibe,a Bent Borg Jensena Department of Animal Science, Aarhus University, Tjele, Denmarka; Center for Geomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmarkb
The butyrate-producing Megasphaera spp. predominate in the pig hindgut and may play important roles in gut health. More- over, one Megasphaera isolate has been reported to produce the boar taint compound, skatole. Here, we provide a 2.58-Mbp draft genome of a pig hindgut isolate, Megasphaera sp. DJF_B143, unable to produce skatole. Downloaded from
Received 4 January 2016 Accepted 8 January 2016 Published 25 February 2016 Citation Li X, Marshall IPG, Schreiber L, Højberg O, Canibe N, Jensen BB. 2016. Draft genome sequence of Megasphaera sp. strain DJF_B143, an isolate from pig hindgut unable to produce skatole. Genome Announc 4(1):e00007-16. doi:10.1128/genomeA.00007-16. Copyright © 2016 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Xiaoqiong Li, [email protected]. http://genomea.asm.org/
acteria of the genus Megasphaera are normal inhabitants of contigs (ca. 1.38 million read pairs; Ϸ0.53 Gbp) were extracted Bthe gastrointestinal tract of mammals (1–4) and can consti- using BBMap version 34.94 (http://sourceforge.net/projects tute a considerable proportion of the microbial community in /bbmap/files) with the “minid ϭ 0.98” option. The extracted reads intestinal contents of pigs (5, 6). Due to its capability to convert were assembled using SPAdes as before, generating 32 contigs lactate to butyrate, the type species M. elsdenii is a probiotic can- (Ͼ200 bp) with ca. 204ϫ coverage. The draft genome sequence of didate for animals (7). Additionally, Megasphaera sp. TrE9262, DJF_B143 has a total length of 2,581,251 bp, an average GϩC isolated from sheep, has been reported to produce skatole (3- content of 49.5%, and an N50 length of 161,321 bp. CheckM ver- methylindole) (8), a main contributing compound of boar taint sion 1.0.3 (13) estimated the genome to be 99.9% complete when
(9). We have isolated strain DJF_B143 belonging to the genus using the gene marker set for the genus Megasphaera. on September 5, 2016 by Aarhus Univ Megasphaera from pig hindgut content. According to 16S Prokka version 1.1 (14) identified 2,297 protein-coding se- rRNA gene sequence analysis, strain DJF_B143 (EU728714) is quences, 9 rRNAs (including 7 5S, 1 16S, and 1 23S) and 56 tRNAs. only distantly related to the described Megasphaera spp., like The absence of an aliphatic amidase (EC 3.5.1.4) gene in M. indica (HM990964, 94.0% identity) and M. micronuciformis DJF_B143 suggests that this strain lacks the metabolic pathway (GU470904, 93.9% identity), and its closest phylogenetic relative from tryptophan to skatole via indole-3-acetate (15). Strain is Megasphaera sp. TrE9262 (DQ278866, 99.9% identity). We DJF_B143 possesses the genomic potential to produce butyrate confirmed the production of butyrate, but observed no skatole from acetate via butyryl coenzyme A (CoA):acetate-CoA trans- production by DJF_B143 either in modified peptone-yeast- ferase (But) (EC 2.8.3.8). glucose or in colon fluid-glucose-cellobiose-agar (CGCA) media. Nucleotide sequence accession numbers. The Megasphaera We sequenced strain DJF_B143 to explore the genomic basis for sp. DJF_B143 genome sequence has been deposited at DDBJ/ these metabolic observations. EMBL/GenBank under the accession number LODR00000000. Megasphaera. sp. DJF_B143 was grown anaerobically in CGCA The version described in this paper is the first version, at 37°C. Genomic DNA was isolated using a Maxwell 16S DNA LODR01000000. purification kit (Promega, USA), and automated DNA purifica- tion was performed on a Maxwell 16 Instrument (Promega). A ACKNOWLEDGMENTS sequencing library was prepared using the Nextera XT kit (Illu- This work was supported by the Danish AgriFish Agency, Ministry of mina, USA). Genome sequencing was performed using the Illu- Food, Agriculture and Fisheries (Project 3405-10-OP-00134). Sequencing mina MiSeq platform with a paired-end 300-bp MiSeq reagent was funded by Aarhus University’s Graduate School of Science and Tech- kit version 3. The resulting sequence reads (ca. 1.7 million read nology and the Section for Microbiology. L.X. was supported by a schol- pairs; Ϸ1 Gbp) were inspected for data quality using FastQC ver- arship from the China Scholarship Council (CSC). sion 0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects We thank Britta Poulsen for skillful technical assistance. /fastqc). Reads were trimmed using Trimmomatic version 0.32 (10) with the following parameters: CROP:250, HEADCROP:20, FUNDING INFORMATION SLIDINGWINDOW:4:20, and ILLUMINACLIP:adapters.fasta:2: China Scholarship Council (CSC) provided funding to Xiaoqiong Li. Aar- 40:15 MINLEN:100. Trimmed Reads were assembled using hus Universitet (AU) provided funding to Ian P. G. Marshall and Lars SPAdes version 3.6.1 (11) with the following parameters: -k Schreiber. 21,33,55,77,99,127 –careful. Contigs most likely originating from The work was supported by the Danish AgriFish Agency, Ministry of contamination were removed using MetaWatt version 3.5 (12). Food, Agriculture and Fisheries (Project 3405-10-OP-00134). Sequencing The decontaminated reads mapping to the retained Megasphaera was funded by Aarhus University’s Graduate School of Science and Tech-
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nology and the Section for Microbiology. The first author was supported compounds by rumen bacteria isolated from grazing ruminants. J Appl by a scholarship from China Scholarship Council (CSC). Microbiol 100:1261–1271. http://dx.doi.org/10.1111/j.1365 -2672.2006.02896.x. REFERENCES 9. Jensen MT, Cox RP, Jensen BB. 1995. 3-Methylindole (skatole) and 1. Counotte GHM, Prins RA, Janssen RHAM, deBie MJA. 1981. Role of indole production by mixed populations of pig fecal bacteria. Appl Envi- Megasphaera elsdenii in the fermentation of DL-[2-13C]lactate in the ru- ron Microbiol 61:3180–3184. men of dairy cattle. Appl Environ Microbiol 42:649–655. 10. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer 2. Elsden SR, Volcani BE, Gilchrist FMC, Lewis D. 1956. Properties of a for Illumina sequence data. Bioinformatics 30:2114–2120. http:// fatty acid forming organisms isolated from the rumen of sheep. J Bacteriol dx.doi.org/10.1093/bioinformatics/btu170. 72:681–689. 11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov 3. Shetty SA, Marathe NP, Lanjekar V, Ranade D, Shouche YS. 2013. AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Comparative genome analysis of Megasphaera sp. reveals niche specializa- Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. tion and its potential role in the human gut. PLoS One 8:e79353. http:// SPAdes: A new genome assembly algorithm and its applications to single- dx.doi.org/10.1371/journal.pone.0079353. cell sequencing. J Comput Biol 19:455–477. http://dx.doi.org/10.1089/ Downloaded from 4. Lanjekar VB, Marathe NP, Ramana VV, Shouche YS, Ranade DR. 2014. cmb.2012.0021. Megasphaera indica sp. nov., an obligate anaerobic bacteria isolated from 12. Strous M, Kraft B, Bisdorf R, Tegetmeyer HE. 2012. The binning of human faeces. Int J Syst Evol Microbiol 64:2250–2256. http://dx.doi.org/ metagenomic contigs for microbial physiology of mixed cultures. Front 10.1099/ijs.0.059816-0. Microbiol 3:410. http://dx.doi.org/10.3389/fmicb.2012.00410. 5. Stanton TB, McDowall JS, Rasmussen MA. 2004. Diverse tetracycline 13. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. resistance genotypes of Megasphaera elsdenii strains selectively cultured 2015. CheckM: assessing the quality of microbial genomes recovered from from swine feces. Appl Environ Microbiol 70:3754–3757. http:// dx.doi.org/10.1128/AEM.70.6.3754-3757.2004. isolates, single cells, and metagenomes. Genome Res 25:1043–1055. http:// 6. Slifierz MJ, Friendship RM, Weese JS. 2015. Longitudinal study of the dx.doi.org/10.1101/gr.186072.114. early-life fecal and nasal microbiotas of the domestic pig. BMC Microbiol 14. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin- http://genomea.asm.org/ 15:184. http://dx.doi.org/10.1186/s12866-015-0512-7. formatics 30:2068–2069. http://dx.doi.org/10.1093/bioinformatics/ 7. Aikman PC, Henning PH, Humphries DJ, Horn CH. 2011. Rumen pH btu153. and fermentation characteristics in dairy cows supplemented with Megas- 15. Whitehead TR, Price NP, Drake HL, Cotta MA. 2008. Catabolic pathway phaera elsdenii NCIMB 41125 in early lactation. J Dairy Sci 94:2840–2849. for the production of skatole and indoleacetic acid by the acetogen Clos- http://dx.doi.org/10.3168/jds.2010-3783. tridium drakei, Clostridium scatologenes, and swine manure. Appl Environ 8. Attwood G, Li D, Pacheco D, Tavendale M. 2006. Production of indolic Microbiol 74:1950–1953. http://dx.doi.org/10.1128/AEM.02458-07. on September 5, 2016 by Aarhus Univ
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49 8. Paper IV
Development of a species-specific Taqman-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet
Xiaoqiong Li, Bent Borg Jensen, Ole Højberg, Samantha Joan Noel, Nuria Canibe
Planned to be submitted to Applied Microbiology Biotechnology
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Development of a species-specific Taqman-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet
Xiaoqiong Li *, Bent Borg Jensen, Ole Højberg, Samantha Joan Noel, Nuria Canibe
Department of Animal Science, Faculty of Science and Technology, Aarhus University, Blichers Allé, P. O. Box 50, DK-8830, Tjele, Denmark
Corresponding author: * Xiaoqiong Li
Tel.: +45 87154257 Fax: +45 8715 0201; E-mail: [email protected]
Key words Chicory root · MGB probe · Olsenella scatoligenes · qPCR · skatole · Taqman
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Abstract
Olsenella (O.) scatoligenes is the only skatole-producing bacterium isolated from the pig gut. Skatole produced from microbial degradation of L-tryptophan, is the main causer of boar taint, an off-odor and off-flavor taint released upon heating meat from some entire male pigs. An appropriate method for quantifying O. scatoligenes could help establishing the relationship between O. scatoligenes abundance and skatole concentration in the pig gut. This study aimed at developing a TaqMan-MGB probe-based, species-specific qPCR assay for rapid quantification of O. scatoligenes. Use of a MGB probe allowed discriminating O. scatoligenes from other closely related species. Moreover, the assay was possible to quantify a minimum of three target gene copies per PCR reaction using genomic DNA-constructed standards, or 1.5×103 cells/g gut digesta using cell-spiked samples as reference standards. The assay was applied to assess the impact of dietary chicory roots on O. scatoligenes population in the hindgut of pigs. O. scatoligenes made up less than 0.01% of the microbial population in the pig hindgut. Interestingly, the highest number of O. scatoligenes was found in young entire male pigs fed high levels of chicory roots. This indicated that the known effect of chicory roots for reducing skatole production was not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Thus, the abundance of O. scatoligenes in the hindgut is not an appropriate indicator of boar taint. Our study is the first one to describe a Taqman-MGB probe qPCR assay for detection and quantification of O. scatoligenes in pigs.
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Introduction
Skatole (3-methylindole) is the main compounds responsible for boar taint, which is an off-odor and off-flavor meat trait released upon heating of meat from some male pigs (Wesoly and Weiler 2012). Boar tainted meat is disagreeable to most consumers and has subsequent negative economic impacts for the meat industry (Jensen et al. 2014). Skatole is produced by anaerobic microbial degradation of L-tryptophan (TRP) via decarboxylation of indol-3-acetic acid (IAA) in the hindgut of pigs. It is produced in both sexes, but increased in some entire male pigs probably due to a reduced skatole metabolism in the liver of these animals (Babol et al. 1999; Zamaratskaia et al. 2004). Supplementation of easily fermentable carbohydrates with low ileal digestibility has been shown to reduce skatole production in the hindgut (Jensen et al. 1995; Knarreborg et al. 2002; Rideout et al. 2004; Lösel and Claus 2005; Hansen et al. 2006; Øverland et al. 2011; Vhile et al. 2012; Zammerini et al. 2012). The most effective carbohydrate seems to be purified inulin or inulin rich feed components like chicory root or Jerusalem artichoke (Jensen et al. 2014).
Whereas many bacteria are able to deaminate TRP to IAA (Patten et al. 2013), only few bacteria have been reported to catalyze the step from IAA to skatole. Clostridium (C.) scatologenes and C. drakei SL1, isolated from soil and acidic sediment, respectively, are able to produce skatole from TRP via IAA (Whitehead et al. 2008), while Olsenella (O.) uli and O. scatoligenes have been shown to produce skatole from IAA, but are unable to convert TRP to skatole (Li et al. 2015). It has been estimated that skatole-producing bacteria represent less than 0.01% of the total intestinal microbiota of the pig (Jensen and Jensen 1998). Of the skatole-producing bacteria, only O. scatoligenes has so far been isolated from the pig gastrointestinal (GI)-tract (Li et al., 2015). Other studies have revealed that dietary fiber enhances Olsenella abundance in pigs (Haenen et al. 2013) and in mice (Mao et al. 2015). However, whether the impact of chicory root on skatole production is related to a change in the population of O. scatoligenes in the GI-tract of pigs has not been elucidated.
Boar taint management strategies require not only detection and identification of skatole-producing bacteria, but also establishment of their density related to skatole production in the hindgut and changes due to dietary fermentable carbohydrates. This paper describes a TaqMan minor groove binder (MGB) qPCR assay targeting the 16S rRNA gene of O. scatoligenes SK9K4T to quantify the O. scatoligenes species. The specificity, sensitivity, and stability of this method were validated from standard DNA
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curves. The established TaqMan-MGB qPCR assay was applied to detect and compare O. scatoligenes densities in the hindgut of pigs fed diets with or without chicory roots.
Materials and Methods
Ethics statement
All animal experimental procedures were carried out in accordance with the Danish Ministry of Justice, Law no. 253 of March 2013 concerning experiments with animals and care of experimental animals. The study was approved by the Danish Animal Experimentation Board (license no. 2010/561-1914).
Experimental protocol and sampling
From an initial total of 36 pigs used in another study, 24 [Duroc × (Danish Landrace × Yorkshire)] pigs with an initial body weight of ~25 kg and aged 7 weeks (13 entire males and 11 females) were included in the current study. Pigs were fed either a standard Danish grower diet based on wheat, barley, and soybean meal (Control); or the same diet in which part of the wheat was substituted with 25% chicory root (Chicory). The composition and chemical analyses of the diets are shown in supplementary material Table 1. Pigs (n=18 in each treatment) were housed in six pens (three pens with six animals per treatment) with partially slatted concreate floors and a few handfuls of woodchips in the resting area. Pigs were allowed ad libitum access to the feed and water. After five weeks on the experimental diets and an average bodyweight of ~60 kg, two pigs from each pen were sacrificed with a captive bolt pistol followed by exsanguination. Immediately after, the large intestine was removed and divided in five sections: cecum, and four equally long segments of the colon including the rectum. Approximately 5 g digesta from the cecum and the third segment of the colon were collected, and aliquots immediately frozen and stored at -20 °C for analyses. After four more weeks on the diet, another two pigs from each pen were killed. Samples from these pigs are not included in the present study. The remaining pigs (2 per pen, 6 per treatment) continued on the same feed for another four weeks, reaching a body weight of ~120 kg, and killed and sampled following the same procedure as described for those killed at ~60 kg body weight.
Extraction of DNA from digesta
Cecal digesta samples were freeze dried at -55°C for 72 hours (Scanvac Coolsafe, model no. 55-4,
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Labogene, Denmark) prior to DNA extraction. Microbial DNA from approximate 200 mg colonic digesta and 50 mg freeze-dried cecal digesta (equal to around 500 mg wet weight) was extracted using the QIAamp® Fast DNA Stool mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for pathogen detection. Briefly, digeta samples were homogenized in the buffer InhibitEX and heated at 95 °C for 5 min to lyse bacterial cells. The lysates were treated with proteinase K and buffer AL at 70 °C for 10 min to remove protein and polysaccharides. DNA was precipitated by ethanol, applied to a column provided in the kit followed by washes with buffers AW1 and AW2, and then dissolved in buffer AE and stored at -20°C.
Bacteria strains, growth conditions and genome DNA extraction
The type strain of O. scatoligenes, SK9K4 DSM 28304T served as a positive control. The type strains of four closely related species: O. uli DSM 7084T, O. profusa DSM 13989T, O. umbonata DSM 22620T, and Atopobium (A.) parvulum DSM 20469T, which were obtained from German Collection of Microorganisms and Cell Cultures (DSMZ), were used as negative controls. Moreover, strains dominating in the pig gut collected in our laboratory: Bacteroides sp. DJF_B097, Lactobacillus sp. DJF_B156, Ruminococcus sp. DJF_VR87, Enterococcus faecalis DJF_O03, Megasphaera elsdenii DJF_RP06, Prevotellaceae bacterium DJF_LS10 and Roseburia sp. DJF_RR73 were also used as negative controls. All strains were cultured anaerobically in modified Peptone-Yeast extract with Glucose medium (PYG-mod) for 48 hours at 37 ˚C as described previously (Li et al., 2015). Genomic DNA (gDNA) from the different reference strains was extracted using a Maxwell 16S DNA purification kit (Promega, Madison, Wisconsin, USA) and automated DNA purification was performed on a Maxwell 16 Instrument (Promega) according to the technical manual provided by the manufacturer.
DNA yield and quality
Quantity and quality of DNA extracted from both digesta samples and pure culture samples was measured by reading the whole absorption spectrum (220–750 nm) with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA quality assessed using absorbance ratio at both 260/280 and 230/260 nm. The DNA concentration in each sample was then quantified with the Qubit fluorometer 3.0 (Life Technologies, Grand Island, NY, USA). The instrument was calibrated with the Qubit® dsDNA HS Assay kit (accurate for initial sample 55
concentration between 10 pg/µL to 100 ng/µL) according to the manufacturer's instructions (Simbolo et al. 2013).
Species-specific primer and probe design
AlleleID® 6.0 (Premier Biosoft, Palo Alto, CA, USA) and the Primer Express Software 3.0 (Life Technologies) were combined to design a primer-probe pair specific for O. scatoligenes SK9K4T targeting the hypervariable region V6 of the 16S ribosomal RNA gene sequence (Chakravorty et al. 2007). Figure 1 shows the sequence targeted by the O. scatoligenes-specific TaqMan MGB probe and primers, and part of homologous 16S rRNA gene sequences from closely related Olsenella species and A. parvulum species. The Basic Local Alignment Search Tool (BLASTn) (http://www.ncbi.nlm.nih.gov/BLAST) and the Ribosomal Database Project (RDP) (https://rdp.cme.msu.edu/probematch/search.jsp) were used for preliminary assessments of oligonucleotide specificity prior to synthesis. The designed primers OscF: 5'-CTTACCAGGGCTTGACATCTTGG-3' (positions: 949-971) and OscR: 5' -ACGACACGAGCTGACGACAG -3' (positions: 1043-1024) were obtained from DNA Technology A/S (Denmark). The TaqMan probe was labeled with 6-carboxyfluorescein (FAM) at the 5' end and a nonfluorescent quencher (NFQ) with MGB ligands was used as the quencher at the 3' end. OscPR: 5'-6-FAM-ACCTGTCTTGGCTCCT-MGB-NFQ-3' (positions: 1014-999) was purchased from Applied Biosystems (Life Technologies, UK).
PCR procedure
To validate each new set of primers, a conventional PCR was performed followed by SYBR Green qPCR. The optimal primer pairs were then selected based on specificity and efficiency of amplification. Next, the TaqMan qPCR was carried out with the primer-MGB probe pair.
PCR was performed on a DOPPIO thermocycler (VWR International, USA) with a PCR program: 94 ˚C for 10 min, denaturation at 94 ˚C for 30 s, annealing at 62 ˚C for 30 s, and elongation at 72 ˚C for 30 s. Thirty cycles were conducted, followed by a final elongation step at 72 ˚C for 10 min. The mix consisted 0.5 µL of the forward primer and the reverse primer, each in a concentration of 10 nmol/µL, 1 µL dNTP (5 nmol/µL), 0.5 µL DyNazymeTM DNA polymerase (2 U/µL), 2.5 µL DyNazyme 10 × buffer (Finnzymes, Finland) and 2 µL template DNA to a final volume of 25 µL per reaction. The
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specificity of the primer pairs was confirmed by attempting to amplify an extensive set of closely related species and pig predominant gut bacterial species as controls. The PCR products were subjected to electrophoresis on a 1.5 % agarose gel. DNA was stained with GelRedTM Nnucleic Acid Gel Stain (Biotium, USA) and viewed under long-wavelength UV light.
Quantitative real-time PCR was performed using a ViiATM 7 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) associated with the ViiA™ 7 RUO software version 1.2.1 (Life Technologies). For SYBR Green qPCR, all PCR experiments were carried out in duplicate with a reaction volume of 10 µL, consisting of 5 µL of 2× Maxima SYBR Green/ROX qPCR master mix (Thermo Fisher Scientific), 300 nM of each primer, and 2 µL of DNA template. The qPCR program was as follows: 50 °C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 62°C for 30 s, and 72°C for 30 s. The same set of reference strains was used as a negative control. A non-template control of nuclease-free water (NTC) was included in each run. To determine the specificity of PCR reactions, melt curve analysis was carried out after amplification.
For the Taqman qPCR, amplifications were carried out in triplicate in a total volume of 10 µL consisting of 5.0 µL TaqMan Universal master mix (Applied Biosystems, Foster City, CA, USA), 300 nM of each primer, and 200 nM of TaqMan MGB probe and 2 µL of gDNA template. For digesta samples, about 10 ng DNA measured by Qubit® fluorometer was used. The qPCR program was: hold 2 min at 50 °C, followed by 10 min at 95 °C, then 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. The same extensive set of reference strains was used as a negative control. Every qPCR run included a negative control and a standard curve consisting of SK9K4T gDNA with known concentrations.
Preparation of PCR standards for Olsenella scatoligenes gDNA standard
A ten-fold dilution series from 3×100 to 3×107 copies per reaction of strain SK9K4T gDNA was set up to generate gDNA standards for absolute quantification of O. scatoligenes. The mass of SK9K4T genome (m) was calculated with the following formula (m = (n × M)/NA) (http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf), in which n is the genome size 23 (bp), NA is Avogadro's number (6.023 × 10 bp/mol), and M is the average molecular weight of a double-stranded DNA molecule (660 g/mol). The genome size of the SK9K4T as determined by the
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high-throughput sequencing Illumina HiSeq 2000 platform (Beijing Genomics Institute (BGI), Shenzhen, China) was 2.47 Mbp. There is only one copy of the 16s rRNA gene located in the chromosome of SK9K4T (Li et al. 2016a). Therefore, it was assumed that one DNA copy is equivalent to one bacterial cell. SK9K4T gDNA extractions were measured by the Qubit® fluorometer to determine the appropriate amount (40.65 ng/uL equivalent to 1.5×107 copies per µL) needed for the initial dilution steps. To generate standard curves, the CT values were plotted against the logarithm of the corresponding template copy numbers. Each standard curve was generated by linear regression of the plotted points. PCR amplification efficiency (E) was calculated from the slope of the standard curve: E =10(−1/slope) −1. The limit of quantification (LOQ) was obtained from gDNA standard curves.
Reference standard
Reference standard curves based on a serial dilution of DNA after extraction from 200 mg of cecal or colonic digesta spiked with 1.5 ×1010 SK9K4T cells/g were constructed (ranging from 3×107 to 3×100 cells per reaction). The initial pure culture stock of SK9K4T was prepared by concentrating a 48-hour-old culture in physiological salt solution. The cell number of the stock (6.0×1010 cells/mL) was determined by microscopic cell counting with the Petroff-Hausser Counting Chamber. The LOQ in intestinal content was obtained from reference standard curves.
MBG TaqMan real-time PCR reproducibility
The reproducibility of the assay was demonstrated by evaluating the variability of the CT values obtained after amplification of 10-fold dilutions of the SK9K4T gDNA standards ranging from 3×100 to 3×107 copies per reaction in triplicate intra- and inter-run. The coefficient of variation (CV) was determined for each of the concentrations of DNA.
DNA recovery assay
For digesta samples, the efficiency of DNA extraction was determined by DNA recovery rate and PCR inhibitor reduction during DNA extraction. The recovery rate was assessed by adding quantified reference strain O.scatoligenes SK9K4T to colonic digesta samples. Specifically, 200 mg colonic digesta samples were spiked with 3×102 to 3×109 cells of SK9K4T, giving final spiked concentrations of 1.5×103 to 1.5×1010 cells/g digesta. The DNA recovery rate of extraction from the digesta samples was assessed
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by first using the gDNA standard curve to convert the Ct to O.scatoligenes genomic copies/reaction, then back-calculating with volumes and dilutions to determine O.scatoligenes cells/reaction for the spiked gut digesta samples. Finally, recovery rate was calculated as the quotient of recovered copies and spiked cells as described by Wattanaphansak et al. (2010).
Quantification of Olsenella scatoligenes and skatole production in gut digesta samples
To illustrate the quantification method, samples from a study on the effect of chicory root on skatole production in the hindgut of entire male and female pigs at two different ages were used. To determine and compare O. scatoligenes numbers in different treatment groups, all real-time PCR data were calculated against reference standards, and log O. scatoligenes cells/g digesta (wet weight) were calculated and presented. The microbial DNA inputs were normalized according to the DNA concentration determined by Qubit® fluorometer to ensure that the O. scatoligenes cells in digesta were compared at the same DNA extraction level during the experiment. The concentration of phenolic- and indolic compounds in digesta was analyzed by high performance liquid chromatography (HPLC) as described by Knarreborg et al. (2002).
Data analysis
The SAS statistical software package, version 9.3 (SAS Institute, Inc., Cary, NC), was used for statistical analyses. Data were analyzed using the software's GLM procedure. The statistical model included the fixed effects of diet, sex, age, and their interactions. Differences between least square means were compared using Tukey-Kramer test (Littell et al. 2002). A P value of <0.05 was considered statistically significant.
Results
DNA yield and purity
Both NanoDrop® spectrophotomer and Qubit® fluorometer were used to measure the amount of extracted DNA. The DNA quality and purity was assessed spectrophotometrically. The A260/A280 ratio was good in most of the extracts (1.8–2.0). DNA concentrations measured by NanoDrop® were higher than those measured by Qubit® (Table 1). Measured by NanoDrop®, cecal and colonic digesta DNA extraction using QIAamp® Fast DNA Stool mini Kit yielded an average concentration of DNA of
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20.9 ± 8.4 µg/g and 58.7 ± 13.2 µg/g wet weight digesta, respectively. The same DNA samples quantified with the Qubit® system showed somehow lower DNA concentrations (i.e. average DNA concentrations of 1.0 ± 0.8 µg/g and 5.1 ± 2.1 µg/g from cecal and colonic digesta, respectively). According to Simbolo et al. (2013), Qubit proves highly reproducible, showing consistent results, and highly correlated to qPCR measurements. Thus, DNA concentrations read from Qubit were used to normalize DNA template concentration in the TaqMan real-time PCR studies.
Specificity of TaqMan-MGB qPCR for Olsenella scatoligenes
The specificity of the primer pairs and the TaqMan-MBG probe was first assessed by BLASTn and the RDP database. A probe match search in the RDP database showed that the sequence of MGB OscP-1014 targeting the V6 area of 16S rRNA matched exactly and only with O. scatoligenes species (including the strains O. sp. SK9K4 (JX905358), O. sp. BS-3 (GU045476), bacterium OL-1 (LK021119), O. sp. J21 (DQ168838) and several clones). Furthermore, it was found that the MGB probe had one mismatch with O. profuse, two mismatches with O. uli and A. parvulum, and three mismatches with O. umbonata (Fig. 1). The specificity of the primers was tested against O. profusa, O. uli, O. umbonata, A. parvulum, and a set of seven dominant pig gut microbial strains using either conventional PCR (Fig. 2A) or SYBR real-time PCR (Fig. 2B). The gDNA from O. scatoligenes, O. profusa, and O. uli was amplified in conventional PCR and SYBR Green real time PCR with expected size of 95 bp. gDNA from the other bacteria strains was not amplified. As shown in Fig. 2C, use of the primer-MGB probe pair in TaqMan MGB real time PCR was specific for the detection of O. scatologens as it did not cross-react with gDNA from any of the other Olsenella species or the gastrointestinal isolates. The Ct difference between O. scatoligenes and non-target samples was equivalent to at least 2 logs of cell numbers. gDNA standard
Dilutions (ranging from 3×100 to 3×106 copies per reaction) of purified gDNA from SK9K4T were used to construct standard curves and assess the probe sensitivity. The Ct values of the qPCR assay with MGB probe ranged between 13.26 (3×106 copies) and 33.93 (3×100 copies) (Table 2). The mean and standard deviation of efficiency of gDNA standard was 95.4 ± 0.5%, the R2 was 1.000 ± 0.000, and the slope was -3.437 ± 0.014. A representative set of gDNA standard curve is shown in Fig.3. The regression line for the curve was y = -3.421x + 35.591. The LOQ giving a reliable and reproducible signal was 60
found to be 4.07 fg, which corresponds approximately to 3 genomic copies per reaction. The gDNA standard curve was used to obtain an absolute quantification of O. scatoligenes (Table 4).
Reference standard
The reference standards based on a dilution series of gDNA from cecal or colonic digesta spiked with 1.5 ×1010 cells/g O. scatoligenes SK9K4T (ranging from 3×101 to 3×107 cells per reaction) showed a log-linear correction of 7 log units (Fig. 4). The regression lines for the reference standard curves of cecal and colonic samples were y = -3.432x + 38.511 (Fig. 4b) and y= -3.471 + 39.612 (Fig. 4D), respectively. The R2 values of the regression lines were 0.998 (cecal samples) and 0.992 (colonic samples), and the amplification efficiencies determined from the linear regression curves were 95.6% and 94.1%, respectively. The LOQ for both cecal and colonical samples was 1.5×104 cells/g (3×101 cells per reaction) when using 10 ng of DNA extract in the real-rime PCR assay.
Reproducibility
The reproducibility of the O. scatoligenes TaqMan qPCR assay was demonstrated by evaluating the variability of the Ct values obtained after amplification of 10-fold serial dilutions of the gDNA standard ranging from 3×106 to 3×100 copies per PCR reaction in triplicate intra- and inter-runs (Table 2). The CV of the mean Ct values obtained for the gDNA standard curve ranged from 0.19% to 1.49% intra-assay and 0.56% to 2.40% inter-assay. The results demonstrated that the established TaqMan-MGB qPCR method was able to characterized O. scatoligenes within a wide range (7 log) with high precision.
Spike recovery
The recovery of O. scatoligenes gDNA was measured on colonic digesta samples from three pigs spiked with a known amount of the reference strain (from 3.0×104 to 3.0×107 cells per PCR reaction). After DNA isolation, quantification, and calculation, recovery rate was found to be between 6.4% and 9.5% (Table 3). The recovery rate of lower amounts (≤ 3.0×103 cells per PCR reaction) of spiked O. scatoligenes gDNA was artificially elevated (data not shown) due to the existence of background (around 105 - 106 O. scatoligenes cells/g) in the colonic samples.
Application of the TaqMan qPCR assay
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The quantification of O. scatoligenes in pig gut digesta samples using qPCR was performed with the reference standards. Generally, higher numerical values of O. scatoligenes were measured in the Chicory group than in the Control group at both ages and in both sexes. However, the results were only significantly different for the young entire male pigs (Table 4). Further, O. scatoligenes number were higher in colonic digesta compared to cecal digesta (6.00 vs. 5.12 log cells/g). Besides, higher numerical values were measured in males compared to females (5.25 vs. 4.92 log cells/g in cecal digesta; and 6.23 vs. 5.96 log cells/g in colonic digesta); and in young pigs compared to old pigs (5.52 vs. 4.71 log cells/g in cecal digesta; and 6.31 vs. 5.89 log cells/g in colonic digesta). Adding chicory roots to the diet significantly reduced (p < 0.001) both skatole and indole concentration in cecal and colonic digesta regardless of sex or age (Table 4).
Discussion
The use of Taqman-MGB qPCR enabled specific enumeration of the skatole-producing bacterium O. scatoligenes in pig hindgut. One of the challenges in the development of qPCR assays is designing primers that specifically target the species of interest in samples containing closely related bacteria. So far, only one 16s RNA gene-based nest PCR protocol has been established to detect Olsenella species, yet this method has failed to discriminate O. uli from O. profusa (Rôças and Siqueira 2005). Our results (Fig 2A and 2B) showed that conventional PCR and SYBR Green qPCR, primer pair alone was not sufficient to distinguish O. scatoligenes from O. profuse and O. uli due to high sequence homology in the variable regions of the 16s rRNA gene among the Olsenella species. Due to significantly improved hybridization properties of MGB probes (Kutyavin 2000), we thus opted to employ a TaqMan-MGB probe. The MGB stabilizes A/T rich duplexes, allowing to use shorter probes with higher melting temperature compared to ordinary DNA probes. The increased specificity of a MGB probe allows discrimination with a single-base mismatch (Van Hoeyveld et al. 2004; Mingxiao et al. 2013). Therefore, in order to increase the specificity of the qPCR reaction for identifying O. scatoligenes, a MGB probe with 1-3 mismatches to the closest species was designed. The Ct difference between O. scatoligenes and the closest species, O. profusa was equivalent to at least two logs of cell numbers. Thus, signals of unspecific targets in gut samples were considered not to compromise the specific enumeration of O. scatoligenes. Our results again showed that MGB probes were more sequence specific than unmodified DNA probes, especially for single-base mismatches.
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Efficiency and accuracy of the qPCR depends on DNA quality. Two main obstacles for good DNA quality are inefficient recovery of total gDNA from the bacterial community and presence of PCR inhibitory compounds from the environmental matrix (Zoetendal et al. 2001). Incomplete lysis of the bacteria in a sample or the presence of inhibitors reduce the number of genomic copies available for PCR or reduce the efficiency of amplification (Coyne et al. 2005). Inhibition of qPCR can be tested by performing a serial dilution. Presence of inhibitory substrates in the digesta samples is possible but in the present study it was considered tolerable, as shown by satisfactory high amplification efficiencies of reference standards, which were around 95%.
The low recovery of DNA was expected and seems impossible to avoid, which was demonstrated by a study using different DNA extraction methods for analysis of cecal microbiota, resulting in 64-99.9% loss of the retrievable DNA (Scupham et al. 2007). The bacterial population of the pig cecum and colon is numerically large with at least 1010 cells/g wet digesta (bacteria is twice as dense in the colon as in the cecum (Butine and Leedle 1989)), resulting in a DNA mass ≥ 32 µg/g wet digesta (calculated from the density of the bacteria, 3Mbp as an average genome size and 1.09 × 10-21 as the mass of DNA per genome). However, in our study, using the Qubit® fluorometer method, only 1.0 and 5.1 µg total gDNA/g were recovered from cecal and colonic digesta, respectively. The low recovery of gDNA extraction was also confirmed by the DNA recovery assay. Less than 10% of O. scatoligenes-specific DNA was detected after DNA isolation and subsequent amplification in our study. In line with our result, (Nathues et al. 2009) reported, using QIAamp® DNA Stool mini Kit, that only 3.5% of the specific DNA added was recovered from spiked feces. Commercial QIAamp® DNA Stool kit has shown high extraction efficiency and PCR-compatibility, and is frequently used in the extraction of gDNA from human feces (McOrist et al. 2002; Nechvatal et al. 2008; Salonen et al. 2010). Contrary to this, another study showed that this kit was less efficient for the extraction of DNA from pig manure samples (Claassen et al. 2013). QIAamp® kit uses a chemical lysis procedure that may lead to incomplete cell lysis. Furthermore, after comparing several DNA extraction methods, FastDNA(™) SPIN Kit for Soil (MP Biomedical) was recommended by Burbach et al. (2016) as a suitable DNA extraction kit for the analyses of porcine gastrointestinal tract samples. The low gDNA recovery in our study may partly be caused by long-term (~ 2 years) storage of digesta samples at -20 ˚C. Degradation of DNA by endonucleases most likely happened during the long-term storage under -20 ˚C, resulting in reduced DNA yield (Metzler-Zebeli et al. 2016). Optimizing sample storage (e.g. stored completely
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desiccated) and DNA extraction methods (e.g. including a step for the mechanical disruption of microbial cells by bead beating) (Yuan et al. 2012) are demanded in future studies.
In qPCR, a plasmid or gDNA carrying the target genes is most commonly used as the standard. This method is relatively straightforward. However, the amplification efficiencies and DNA recovery efficiencies of plasmids/gDNA or cell cultures used for generation of a standard curve may not be similar to that of the DNA extracted from environmental samples. In order to overcome this problem, a known concentration of exogenous cells was spiked into the digesta samples as a reference standard. Since DNA is extracted from a matrix in the presence of a known amount of spiked target microbe as a reference standard, inherent variability in extraction and amplification efficiencies affect both equally (Coyne et al. 2005). To obtain a more precise determination of microbes of interest in a complex sample, there are increasing numbers of studies using cell-spiked samples as reference standards (Abildgaard et al. 2010; Hariganeya et al. 2013; Sattler et al. 2014). According to our current result, applying a gDNA standard to estimate O. scatoligenes in gut samples would lead to a more than 10-fold under-estimation of cell numbers in the gut digesta due to the low DNA recovery efficiency. Therefore, spiked reference standards have to be used for enumeration of O. scatoligenes in gut digesta samples.
In other studies, the standard curves have normally been created by inoculating the complex samples, free of the target microbe, with serially decreasing amounts of the target microbe; obtaining detection limits of around 102-103 (Abildgaard et al. 2010), or 103-104 cells/g (Wattanaphansak et al. 2010). The drawback of this approach is that reference samples are spiked independently, potentially introducing another source of error owing to inconsistent DNA extraction efficiency of these samples (Coyne et al. 2005). In our study, the gut digesta samples were first spiked and then DNA was isolated and serially diluted. Sattler et al. (2014) claimed that this approach may lead to a relatively low detection limit at 102-101 cells/g. Since O. scatoligenes is a common habitant of pig gut microbiota, it is not possible to find gut digesta samples that are O. scatoligenes free. Dilution of the gDNA largerly eliminates the background number of O. scatologenes, thereby reducing the interference of the target DNA during amplification reaction by indigenous O. scatoligenes considerably. The gDNA standard, which was used to obtain an absolute quantification of O. scatoligenes gDNA copies, had a quantification limit of 3 copies per PCR reaction. However, the quantification limit was compromised to 103 cells/g when using reference standards owing to poor DNA recovery efficiency. The PCR could detect the presence of O.
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scatoligenes at 102 cells/g of gut digesta, but was unable to quantify the population accurately or precisely at this low O. scatologenes level, which is a common default of qPCR studies. The qPCR assay had high level of reproducibility, as shown by acceptable low intra-assay and inter-assay coefficients of variation.
Little is known about the presence and identity of skatole-producing bacteria in the hindgut of pigs and how these bacteria are affected by fermentable dietary fibre such as inulin from chicory roots. As revealed by 16s rRNA pyrosequencing studies, Olsenella genus constitutes 0.12% (from 0.02% to 0.94%) of the total microbiota in pig feces (Kim et al. 2011). Using the most probable number technique in combination with skatole measurements, it has been estimated that the population of skatole-producing bacteria accounts for less than 0.01% of the total microbiota in pig fecal samples (Jensen and Jensen 1998). We had screened 122 OTUs, which represent 2678 isolates from the GI-tract of pigs for their ability to produce skatole from TRP or IAA. The results showed that none of them except O. scatoligenes SK9K4 was able to produce skatole (Li et al. 2015). Megasphaera sp. TrE9262 has been reported to produce skatole (Attwood et al. 2006), however, the isolated Megasphaera strain DJF_B143, which has 99.9% 16S rRNA gene identity with strain TrE9262 was tested and was unable to produce skatole in our previous study (Li et al. 2016b). Therefore, we suspected that skatole-producing bacteria were limited only to a few species and O. scatoligenes is the main dominant skatole-producing bacterium in the pig GI-tract. Accordingly, in the current study, we found that O. scatoligenes accounted for less than 0.01% (105 – 106 cells/g) of the total population of the hindgut microbiota, which is in agreement with the finding by Jensen and Jensen (1998). An increased proliferation of Olsenella genus by dietary fiber has been revealed by some studies (Haenen et al. 2013; Mao et al. 2015). Interestingly, in the current study, dietary addition of chicory roots addition only significantly increased the abundance of O. scatoligenes in ~60 kg entire male pigs.
A reduction of skatole production in the pig hindgut by adding the inulin rich feed component chicory root in the diet was anticipated (Wesoly and Weiler 2012; Zammerini et al. 2012), and was proved in the current study. Three main underlying mechanisms behind this effect have been proposed in the literature: i) a high content of fermentable fiber in the hindgut increases the microbial activity, resulting in more tryptophan incorporated as bacterial biomass and thereby leaving less substrate for skatole production (Jensen et al., 1995a , Xu et al., 2002); ii) an increased butyrate production due to inulin-type fructans in the diet (Falony et al., 2006) reduces sloughing of enterocytes and, as such, less
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endogenous tryptophan from cell debris is available for skatole production by microbes (Claus et al., 2003). Both of the two mechanisms would result in a reduction of tryptophan substrate for skatole production, therefore, iii) altered gut microbiota characterized by a lower abundance of skatole-producing bacteria could be expected when adding high levels of dietary fermentable fiber. To our knowledge, this is the first study showing that the skatole reducing effect of chicory roots is not by inhibiting the growth of skatole-producing bacteria growth in the pig hindgut. Contrary to this, chicory roots seems to increased O. scatoligenes proliferation, though this was only significant in young entire male pigs.
In conclusion, this study demonstrated for the first time the reliable and specific identification of O. scatoligenes from pig gut content samples by TaqMan®-MGB real-time PCR on the ViiATM 7 Real-time PCR System. The assay was shown to have high specificity, sensitivity, accuracy, and reproducibility. Meanwhile we proved that the reduction effect of chicory root on skatole production in the hindgut was not associated with a reduced numbers of O. scatoligenes. Thus, the abundance of O. scatoligenes in the hindgut of pigs seems not to be a valid indicator of boar taint.
Acknowledgments
This work was supported by the Danish AgriFish Agency, Ministry of Food, Agriculture and Fisheries (Project 3405-10-OP-00134). L.X. was supported by a scholarship from the China Scholarship Council (CSC). We thank Helle Handll and Kasper Poulsen for skillful technical assistance
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Table 1 Comparison of microbial DNA concentration from pig digesta samples measured by NanoDrop® spectrophotomer and Qubit® fluorometer quantitation platforms.
Sample NanoDrop® (µg/g) Qubit ® (µg/g) Cecal digesta (n=24) 20.9 ± 8.4 1.0 ± 0.8 Colonic digesta (n=47) 58.7 ± 13.2 5.1 ± 2.1
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Table 2 Reproducibility, intra-assay and inter-assay coefficient of variation for TaqMan qPCR.
No. of SK9K4T genome Intra-assay Inter-assay
(copies/reaction) CT value CV (%) CT value CV (%) 3×106 13.30 ± 0.20 1.49 13.26 ± 0.10 0.74 3×105 16.94 ± 0.07 0.42 16.86 ± 0.13 0.80 3×104 20.33 ± 0.04 0.19 20.01 ± 0.48 2.40 3×103 23.73 ± 0.09 0.37 23.62 ± 0.13 0.56 3×102 27.17 ± 0.23 0.85 27.80 ± 0.12 0.58 3×101 30.46 ± 0.32 1.05 30.38 ± 0.22 0.72 3×100 33.93 ± 0.46 0.83 33.73± 0.25 0.73
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Table 3 DNA recovery rates from serially diluted O. scatoligenes SK9K4T cells spiked into colonic digesta
Spikeda Detectedb Recoveryc (cells/reaction) (copies/reaction) (%)
3.0×107 (2.64 ± 0.45) ×106 8.8 ± 1.5 3.0×106 (2.86 ± 0.59) ×105 9.5 ± 2.0
3.0×105 (2.67 ± 0.42) ×104 8.9 ± 1.4 3.0×104 (1.92 ± 0.49) ×103 6.4 ± 1.6 a Three 200 mg colonic digesta samples were spiked with 3×102 to 3×109 cells of SK9K4T, giving spiked concentrations at 3×100 to 3×107 cells per PCR reaction. Samples with values below 3.0×104 cells per PCR reaction are not shown due to abundance of indigenous O. scatoligenes in colon. b Detected O. scatoligenes genomic copies/reaction was obtained from gDNA standard curve. c Recovery rate was calculated as the quotient of recovered copies and spiked cells
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Table 4 Olsenella scatoligenes (log cell/g digesta), skatole (mmol/kg digesta), and indole (mmol/kg digesta) concentrations in cecal and colonic digesta of pigs fed the Control or Chicory1.
Diet2 Control Chicory
Sex Female Male Female Male P-value
Weight (kg) 60 (n=3) 120 (n=2) 60 (n=3) 120 (n=4) 60 (n=2) 120 (n=3) 60 (n=4) 120 (n=3) Diet Sex Weight D*S D*W S*W D*S*W
Caecum
O. scatoligenes 4.84±0.28a 4.74±0.23a 5.02±0.20a 4.73±0.23a 5.38±0.23a 4.61±0.28a 6.77±0.23b 4.74±0.20a 0.005 0.023 <0.001 0.061 0.003 0.047 0.135
Skatole 0.8±0.9 2.4±0.7 2.3±0.6 2.9±0.7 0.0±0.7 0.0±0.9 0.0±0.7 0.0±0.6 0.001 0.360 0.315 0.360 0.315 0.644 0.644
Indole 2.7±1.6 2.9±1.3 5.4±1.6 3.7±1.3 0.7±1.3 1.6±1.6 0.5±1.3 0.4±1.2 0.008 0.584 0.860 0.209 0.573 0.468 0.786
Colon
O.scatoligene 6.01±0.13a 5.76±0.11a 5.93±0.09a 5.83±0.11a 6.12±0.11a 5.97±0.13a 7.23±0.11b 6.02±0.11a <0.001 0.002 <0.001 0.002 0.006 0.010 0.002
Skatole 16.5±5.3 29.4±4.3 21.9±3.7 24.7±4.3 0.0±4.3 1.0±5.3 0.2±4.3 0.2±3.7 <0.001 0.990 0.202 0.917 0.257 0.392 0.473
Indole 6.0±1.9 7.7±1.6 10.1±1.4 5.1±1.6 0.3±1.6 1.2±1.9 0.2±1.6 0.3±1.4 <0.001 0.888 0.610 0.586 0.361 0.120 0.217
1 Values are least square means and standard error.
2 Experimental diets: Control = a standard Danish grower diet based on wheat, barley, and soybean meal; Chicory = the same diet in which barley and wheat had been substituted with 25% chicory root.
a,b Means within a row without a common superscript letter differ (p < 0.05).
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Fig. 1 Sequence targeted by the O. scatoligenes-specific MGB probe and primers, taking into account analogous sequences in the closely related Olsenella species and Atopobium species. Mismatches are marked in red.
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Fig. 2 Specificity of the primer pair alone and TaqMan primer-probe pair for Olsenella scatoligenes SK9K4T using genomic DNA targeting a specific region of the 16s rRNA gene sequence. A) Conventional PCR with the primer pair alone. 1, O. scatoligenes; 2, O. uli; 3, O. profusa; 4-12, Non-specific cultures; 13 distilled water. B) SYBR Green real-time PCR with the primer pair alone. C) TaqMan-MGB real-time PCR with the primer-probe pair.
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Fig. 3 A representative amplification plot (A) and standard curve (B) of O. scatoligenes Taqman-MGB qPCR assay with tenfold dilutions of O. scatoligenes SK9K4T genomic DNA ranging from 3×106 to 3×100 copies per reaction by using ViiA™ 7 RUO software version 1.2.1.
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Fig. 4 Amplification plots (A, C) and standard curves (B, D) of O. scatoligenes Taqman-MGB qPCR assay with tenfold dilutions of DNA from cecal (A, B) or colonic digesta (C, D) spiked with 1.5 ×1010 cells/g O. scatoligenes SK9K4T (ranging from 3×107 to 3×102 cells per reaction) by using ViiA™ 7 RUO software version 1.2.1.
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Supplementary table 1 Ingredients and the analyzed composition of the experimental diets.
Ingredient composition (%) Control/Butyrate Chicory Wheat 55.97 28.84 Barley 20.00 20.00 Dehulled toasted soybean meal 9.93 12.00 Dehulled sunflower cake 8.00 8.00 Dried milled chicory roota 0.00 25.00 Soybean oil 1.70 1.40 Sugar beet molasses 1.50 2.00 Calcium carbonate 1.42 1.33 Monocalcium phosphate 0.36 0.35 Sodium chloride 0.49 0.48 L-Lysine-HCl, 98% 0.32 0.29 DL-Methionine , 98% 0.03 0.04 Threonine, 98% 0.08 0.08 Vitamin and mineral premixb 0.20 0.20 Analyzed composition (g/kg dry matter)
DM 866 778 Fat 34 28 Crude protein 154 157 Ash 42 54 Calcium 6.01 6.65 Phosphorus 4.31 4.13 Valine 7.00 6.79 Cystein + Cystine 3.29 2.88 Lysine 8.88 9.33 Methionine 2.76 2.80 Threonin 5.62 6.16 a The content of fructan in the dried milled chicory root was 65.2 %. b Providing the following per kilogram of diet: 3696 IU of vitamin A; 370 IU of vitamin D3; 61 mg of vitamin E; 1.8 mg of vitamin K3; 1.8 mg of vitamin B1; 1.8 mg of vitamin B2; 2.8 mg of vitamin B6; 0.02 mg of vitamin B12; 9.2 mg of Ca-D-pantothenic acid; 18.5 mg of niacin; 0.05 mg of biotin; 55 mg of DL-alpha-tocopherol; 61 mg of DL-alpha-tocopherol acetate; 74 mg of Fe (Fe (II) sulphate); 13 mg of Cu (Cu(II) sulphate) and 37 mg Mn (Mn(II) oxide).
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9. Paper V
Phylogenetic diversity of cultivable butyrate-producing bacteria from pig gut content and feces
Xiaoqiong Li, Ole Højberg, Nuria Canibe, Bent Borg Jensen
Published in Journal of Animal Science 2016, 94:377–381, doi:10.2527/jas2015-9868
80 Published October 6, 2016
Phylogenetic diversity of cultivable butyrate-producing bacteria from pig gut content and feces
X. Li,*1 O. Højberg,* N. Canibe,* and B. B. Jensen*
*Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark
ABSTRACT: Butyrate is a preferred energy source for appeared to represent novel species. The abundance colonocytes and is considered crucial for maintaining of the cultivable butyrate-producing community was colonic health in humans and animals. To investigate 8.3%, 10.7%, 17.2%, and 7.0% in the ileum, cecum, the diversity of cultivable butyrate-producing bacteria colon, and feces, respectively. Butyrate producers in pig gut, bacteria were isolated from intestinal diges- within clostridial clusters IX (Acidaminococcus and ta (Exp. 1) and feces (Exp. 2) of fnishers. Based on Megasphaera) and XIVa (Coprococcus/Eubacterium/ 16S rRNA gene sequencing, 2,762 isolates were cat- Roseburia) were found to be the most abundant. egorized into 122 operational taxonomic units (OTUs). Members of the Bacteroidetes and Fusobacteria phyla Representative isolates of 31 OTUs produced butyrate. were also identifed as butyrate producers. Notably, Complete 16S rRNA gene sequences of the 31 OTUs high-fber diet was correlated with lower abundance were subjected to phylogenetic analysis, and based on a of Acidaminococcus fermentans, Clostridium perfrin- level of <97% 16S rRNA gene sequence similarity with gens, and Clostridium rectum but higher abundance of their nearest validly named neighbors, 15 of the OTUs the commensal Megasphaera elsdenii. Key words: butyrate, cultivable bacteria, diversity, pigs
© 2016 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2016.94:377–381 doi:10.2527/jas2015-9868 INTRODUCTION wk: a low fber diet (LF), a high fber oat bran diet (OB), or a high fber wheat bran diet (WB). The Butyrate has been identifed as the major energy composition and chemical analyses of the diets are source for colonic epithelial cells and has multiple given by Christensen et al. (1999). Digesta from the benefcial effects on the host (Berni Canani et al., ileum, cecum, and colon were suspended, homoge- 2012). Hence, butyrogenic bacteria are considered nized, diluted, and incubated (Jensen and Jorgensen, promising future probiotics. Despite the likely im- 1994). Experiment 2 included 5 castrated male pigs portance of butyrate-producing bacteria to the host, fed a standard Danish pig diet for 4 wk. Fecal sam- their population structure and taxonomy have not ples were processed as in Exp. 1. been extensively explored in pigs. Isolation and Identifcation of Gut Bacteria MATERIALS AND METHODS From each sample, 50 colonies were picked from the RGCA medium (Exp. 1), and 20 colonies were Animals, Diets, and Sampling picked from each of 11 different media (see Table S1 Bacteria were isolated from two experiments in the supplemental material, found online; Exp. 2). (Exp.) with crossbred (Duroc × (Danish Landrace × Colonies were anaerobically picked and transferred Yorkshire)) pigs aged 4 to 6 mo. Experiment 1 con- to CGCA medium (38°C, 1 wk). Purity of the culture sisted of 12 female pigs from 4 litters. One pig from was confrmed by morphology. This was repeated each litter was fed one of the following diets for 3 until microscopic examination indicated culture pu- rity. Phenotypic characterization in the PYG-mod medium (Li et al., 2015) was used to presumptively 1Corresponding author: [email protected] identify and group the 2,762 isolates.
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Table 1. Characterization of the cultivable butyrate-producing bacteria isolated from pig gut content and feces
1 2 3 DJF Species identifed Organic acid production (mM) Exp. 1 % of isolates Exp. 2 % strain (16S rRNA % homology; accession no.) F A P B L LF OB WB of isolates4 Clostridial cluster XIVa VP48 Eubacterium rectale ATCC 33656T (99.8; NR074634) — — 0.1 8.5 21.6 — — — 0.3 CR35 Eubacterium rectale ATCC 33656T (95.2; NR074634) 11.5 4.8 0.1 6.0 7.3 — — — 0.1 RR13 Roseburia faecis M72/1T (97.7; NR_042832) ———4.6 — — — — 0.1 RR73 Roseburia faecis M72/1T (97.3; NR_042832) 1.1 — 0.1 10.3 14.0 — — — 1.0 CR50 Eubacterium ramulus ATCC 29099T (98.6; L3462316) 10.3 2.0 0.1 9.6 0.2 — — — 0.2 CP64 Coprococcus catus VPI C6–61T (97.3; AB038359) — 0.8 3.3 4.9 — 2.1 0.9 0.3 0.3 VP35 Eubacterium ruminantium GA195T (91.9; NR_024661) 7.2 1.6 — 2.9 29.6 — — — 0.1 B005 Coprococcus eutactus ATCC 27759T (96.7; NR_044049) 3.4 1.8 0.2 1.2 0.9 0.2 — 0.2 — CR49 Coprococcus eutactus ATCC 27759T (98.8; NR_044049) 8.2 3.0 0.7 3.3 34.0 0.3 0.4 0.2 0.1 RP51 Clostridium symbiosum ATCC 14940T (95.0; NR_118730) 6.1 23.6 — 7.8 12.2 — — — 0.2 VP52 Butyrivibrio hungatei JK 615T (91.5; NR_025525) 2.0 9.2 — 6.4 12.4 — — — 0.1 B223 Roseburia faecis M72/1T (91.3; NR_042832) 0.1 — 0.1 4.0 1.2 — 0.4 0.2 — Clostridial cluster XV B256 Anaerofustis stercorihominis WAL 14563T (97.0; NR_027562) — — 0.1 4.6 — — — 0.2 — Clostridial cluster I VP39k1 Clostridium sardiniense DSM 2632T (98.8; NR_112226) 11.8 8.7 — 12.5 12.1 — — — 0.2 B043 Clostridium perfringens JCM 1290T (99.8; NR_113204) 2.8 18.9 2.2 7.8 3.7 2.4 1.2 0.5 — Clostridial cluster IV VR09 Faecalibacterium prausnitzii ATCC 27768T (98.2; NR_028961) 1.1 — 0.1 4.2 1.1 — 0.2 0.2 0.2 VR33k2 Subdoligranulum variabile BI 114T (96.8; NR_028997) 10.2 1.9 — 4.4 — — — — 0.1 CP67 Eubacterium desmolans ATCC 43058T (95.6; NR_044644) — — 0.1 6.8 — — — — 0.1 B152 Eubacterium desmolans ATCC 43058T (94.9; NR_044644) ———5.0 0.1 0.2 — — — VP44 Flavonifractor plautii Prevot S1T (95.5; NR_043142) 5.4 1.0 0.3 21.0 0.1 — — — 0.1 B280 Oscillibacter valericigenes Sjm18–20T (95.1; NR_074793) — — 0.1 3.3 — — — 0.2 — Clostridial cluster IX RP06 Megasphaera elsdenii DSM 20460T (99.5; NR_102980) 4.5 1.7 3.5 7.4 — — 2.3 0.3 2.0 B143 Megasphaera indica NMBHI-10 T (94.0; HM990965) 0.2 0.5 0.2 0.6 — 4.3 0.2 0.2 — RP55 Acidaminococcus fermentans DSM 20731 T (98.1%; NR_074928) 2.6 12.9 0.2 6.0 32.5 7.2 0.5 — 1.8 Clostridial cluster XVI VR85 Holdemanella biforme DSM 3989T (96.6; NR_044731) 0.1 — — 3.2 30.6 — — — 0.1 Unclassifed Clostridium B077 Eubacterium pyruvativorans I-6T (93.0; NR_042074) — 2.7 0.2 0.5 4.9 — — 0.3 — Fusobacteria B254 Fusobacterium perfoetens ATCC 29250T (98.6; NR_044688) — 0.4 0.5 19.8 13.5 — 0.2 1.4 — B100 Clostridium rectum NCIMB 10651T (99.6; X77850) 4.0 0.7 2.1 19.1 0.9 5.0 1.8 1.6 — Bacteroidetes B089 Odoribacter splanchnicus DSM 20712T (99.6; NR_074535) — 17.4 19.6 2.6 0.1 — 0.2 — — B268 Butyricimonas virosa MT12T (98.3; NR_041691) — — 0.1 4.6 — 0.2 — — — B220 Bacteroides plebeius M12T (90.6; AB200217) 0.5 ——4.0 — — 0.2 — — 1Operational taxonomic units (OTUs) which appeared to represent novel species on a level of <97% 16S rRNA gene sequence similarity with their nearest validly named neighbors are in bold. 2Formate (F); acetate (A); propionate (P); butyrate (B); lactate (L); not detected (—). 3Total number of isolates 582, 563, and 577 in diet LF (low fber), OB (oat bran diet), and WB (wheat bran diet), respectively. 4Total number of isolates 1,040.
Metabolic End Product Quantifcation DNA Extraction and 16S Ribosomal RNA Gene Sequencing and Phylogenetic Analysis Isolates were incubated (37°C, 1 wk) in triplicate in Hungate tubes with PYG-mod. Culture superna- DNA of each strain was extracted and purifed, tants were analyzed for organic acids by gas chroma- and the 16S rRNA gene was amplifed by PCR (Li et tography (Li et al., 2015). al., 2015). The near-complete 16S rRNA sequences (Eurofns MWG Biotech) were BLASTed and submit- ted to GenBank (accession numbers shown in Fig. 1).
82 Butyrate-producing bacteria in pigs 379
Figure 1. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of the butyrate-producing pig gut isolates (in bold) falling within clos- tridial clusters. GenBank accession numbers are given in parentheses. Prevotella brevis GA33T was used as the out-group sequence.
83 380 Li et al.
Phylogenetic analysis was conducted as described by Li Acidaminococcus (A.) fermentans and Megasphaera et al. (2015). (M.) elsdenii, within clostridial cluster IX, were the most frequently isolated butyrate producers in our study, ac- RESULTS AND DISCUSSION counting for 2.3% and 1.3% of the isolates, respectively. Megasphaera elsdenii has been shown to be the most dominating butyrate producer isolated from the pig gut Abundance of Butyrate-Producing Bacteria (Levine et al., 2013). Leser et al. (2002) also showed that In Exp. 1, 581, 577, and 564 colonies were picked M. elsdenii comprised up to 1.2% of the pig gut bacteria. from the ileum, cecum, and colon samples, respec- tively. In Exp. 2, 1,040 colonies were picked from 11 Distribution of Butyrate-Producing different media. Based on morphology, glucose fer- Bacteria among Diets mentation, and 16S rRNA gene, the 2,762 isolates were categorized into 122 operational taxonomic units Clostridium perfringens and C. rectum were the (OTUs). Of these, 31 OTUs were identifed to pro- only butyrate-producing bacteria isolated from the il- duce butyrate. In total, 8.3%, 10.7%, 17.2% and 7.0% eum and were most abundant in pigs fed the LF diet butyrate-producing strains were isolated from the il- (Table 1). The type of dietary fber (DF) can affect mi- eum, cecum, colon and feces, respectively. crobial composition and metabolic activity differently. Dietary fber has been reported to have an inhibitory Characterization of Butyrate-Producing Bacteria effect on C. perfringens, which is an important pig and human pathogen. In line with previous studies (Vhile Based on 16S rRNA gene sequence comparison et al., 2012), we detected lower abundance of C. per- (97% identity cutoff), 15 of the 31 OTUs appeared to fringens in pigs fed the high fber diet. In contrast, represent novel species. Twenty-fve OTUs were dis- feeding the OB diet, but not the WB diet, was corre- tributed among 6 known clostridial clusters (12, 1, 2, 6, lated with a higher colonic abundance of M. elsdenii. 3, and 1 OTUs among clusters XIVa, XV, I, IV, IX, and Similar results have been reported for pigs fed a fruc- XVI, respectively). Isolates from the Bacteroidetes tan-rich diet with chicory root (Mølbak et al., 2007). In and Fusobacteria phyla were also identifed as butyr- our study, feeding a LF diet favored Coprococcus ca- ate producers (Table 1 and Fig. 1). tus and A. fermentans growth. The current study sug- Apart from butyrate, formate, acetate, propionate, gests that DF promotes a shift of butyrate-producing and lactate were commonly produced by the butyrate- community in the gut toward more benefcial bacteria producing bacteria (Table 1). The highest butyrate con- (e.g., M. elsdenii), while inhibiting the proliferation of centrations were produced by Fusobacterium perfoe- potential pathogenic bacteria (e.g., A. fermentans, C. tens and Clostridium (C.) rectum and strains distantly perfringens, and C. rectum). related to Flavonifractor plautii. Bacteria belong- ing to clostridial clusters IX (Acidaminococcus and LITERATURE CITED Megasphera) and XIVa (Coprococcus/Eubacterium/ Berni Canani, R., M. Di Costanzo, and L. Leone. 2012. The epi- Roseburia) were the most abundant cultivable butyr- genetic effects of butyrate: Potential therapeutic implications ate producers in pig gut, accounting for 45.4% and for clinical practice. Clin. Epigenetics 4:4. doi:10.1186/1868- 19.3% of the total 280 butyrate-producing isolates and 7083-4-4 4.6% and 2.0% of the total 2,762 isolates, respectively. Christensen, D. N., K. E. B. Knudsen, J. Wolstrup, and B. B. Data from 16S rRNA gene libraries of pig in- Jensen. 1999. Integration of ileum cannulated pigs and in vi- tro fermentation to quantify the effect of diet composition on testinal microbiota have shown that the Roseburia/ the amount of short-chain fatty acids available from fermen- Eubacterium group is common in pig gut, comprising tation in the large intestine. J. Sci. Food Agric. 79:755–762. up to 8% of the total bacterial population (Leser et al., doi:10.1002/(SICI)1097-0010(199904)79:5<755::AID- 2002). Only 0.8% of the bacteria fell within this group JSFA248>3.0.CO;2-2 in our study. Similarly, Faecalibacterium (F.) praus- Jensen, B. B., and H. Jorgensen. 1994. Effect of dietary fber on microbial activity and microbial gas-production in various nitzii has been reported to comprise up to 2.9% of total regions of the gastrointestinal-tract of pigs. Appl. Environ. bacteria in pigs (Leser et al., 2002). However, in the Microbiol. 60:1897–1904. current study, only 0.1% of the isolates was F. praus- Leser, T. D., J. Z. Amenuvor, T. K. Jensen, R. H. Lindecrona, M. nitzii related. Faecalibacterium prausnitzii is known Boye, and K. Moller. 2002. Culture-independent analysis of to be notoriously diffcult to cultivate and preserve. gut bacteria: The pig gastrointestinal tract microbiota revis- ited. Appl. Environ. Microbiol. 68:673–690. doi:10.1128/ The Roseburia/Eubacterium group and F. prausnitzii AEM.68.2.673-690.2002 may thus have been underrepresented in our study.
84 Butyrate-producing bacteria in pigs 381
Levine, U. Y., T. Looft, H. K. Allen, and T. B. Stanton. 2013. Mølbak, L., L. E. Thomsen, T. K. Jensen, K. E. Bach Knudsen, Butyrate-producing bacteria, including mucin degraders, and M. Boye. 2007. Increased amount of Bifdobacterium from the swine intestinal tract. Appl. Environ. Microbiol. thermacidophilum and Megasphaera elsdenii in the colonic 79:3879–3881. doi:10.1128/AEM.00589-13 microbiota of pigs fed a swine dysentery preventive diet con- Li, X., R. L. Jensen, O. Hojberg, N. Canibe, and B. B. Jensen. 2015. taining chicory roots and sweet lupine. J. Appl. Microbiol. Olsenella scatoligenes sp. nov., a 3-methylindole (skatole) 103:1853–1867. doi:10.1111/j.1365-2672.2007.03430.x and 4-methylphenol (p-cresol) producing bacterium isolated Vhile, S. G., N. P. Kjos, H. Sorum, and M. Overland. 2012. Feeding from pig faeces. Int. J. Syst. Evol. Microbiol. 65:1227–1233. Jerusalem artichoke reduced skatole level and changed intes- doi:10.1099/ijs.0.000083 tinal microbiota in the gut of entire male pigs. Animal 6:807– 814. doi:10.1017/S1751731111002138
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9.1 Supplementary Materials for Paper V
Phylogenetic Diversity of Cultivable Butyrate-producing Bacteria from Pig Gut Content and Feces
Xiaoqiong Li1*, Ole Højberg1, Nuria Canibe1, Bent Borg Jensen1
1Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark
Corresponding author: *Xiaoqiong Li
Tel.: +45 87154257 Fax: +45 8715 0201; E-mail: [email protected]
Running title: butyrate-producing bacteria from pig
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Table S1. Non-selective and selective media for isolating anaerobic bacteria from pig gut content and feces.
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10. Paper VI
Effect of chicory roots and exogenous butyrate on skatole production and gut microbiota of entire male pigs
Xiaoqiong Li, Bent Borg Jensen, Nuria Canibe
Planned to be submitted to Applied and Environmental Microbiology
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Effect of chicory roots and exogenous butyrate on skatole production and gut microbiota of entire male pigs
Xiaoqiong Li, Bent Borg Jensen, Nuria Canibe*
Department of Animal Science, Faculty of Science and Technology, Aarhus University, Blichers Allé, P. O. Box 50, DK-8830, Tjele, Denmark
Corresponding author: * Nuria Canibe
Tel.: +45 8715 8058 Fax: +45 8715 4249; E-mail: [email protected]
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Abstract
The effect of high levels of dietary chicory roots (25%) and intracecal exogenous butyrate infusion on skatole formation and gut microbiota was investigated in order to clarify the mechanisms underlying the known reducing effect of chicory roots on skatole production in entire male pigs. A Latin square experimental design with 3 treatments (Control, Chicory and Butyrate), 3 periods, and 6 animals, resulting in 2 animals per treatment per period, was carried out. Chicory roots, as expected, showed the lowest numerical levels of skatole in both feces and plasma. Butyrate infusion resulted in the highest levels of skatole in feces and in plasma. The impact of the treatments on microbiota composition was demonstrated by an increased abundance of the skatole-producing bacterium Olsenella scatoligenes in the Chicory group compared to the Control group (p = 0.06), and a numerically higher relative abundance of Olsenella compared to the Control and Butyrate group. Regarding butyrate-producing bacteria, the Chicory group had lower abundance of Roseburia, but a numerically higher abundance of Megasphaera compared to the Control group. Parameters of alpha and beta diversity analyses showed significant differences between the Chicory and the Butyrate groups. Lower species richness was found in the Chicory group compared to the Butyrate group. Besides, beta diversity revealed that the Chicory group formed a distinct cluster, whereas the Control and Butyrate groups clustered more closely to each other. The data indicated that the skatole-reducing effect of chicory root is neither via inhibition of cell apoptosis by butyrate, nor via suppression of skatole-producing bacteria in the pig hindgut. Thus, the mode of action is most likely through increased microbial activity with a corresponding high incorporation of amino acids into bacterial biomass, and thereby suppressed proteolytic conversion of tryptophan into skatole, as indicated in the literature.
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Introduction
Boar taint is an offensive odor and flavor released upon heating meat from some pubertal or sexually mature entire male pigs, making the meat undesirable for human consumption, with the subsequent negative economic impact for the pig industry (1). Skatole (3-methylindole), which has a fecal-like odor, is produced by microbial degradation of L-tryptophan (TRP) in the hindgut of pigs, and is together with androstenone, the main compound responsible for boar taint (2). Skatole production is limited to a few bacterial species, of which, only one species, Olsenella (O.) scatoligenes, has been isolated from the pig gut so far (3). Skatole is produced in the hindgut of both pig sexes; however, higher concentrations are measured in the back fat of entire male pigs, probably due to reduced skatole metabolism in the liver (4–6).
Surgical castration of male pigs within the first week of life is a common practice in many countries to prevent boar taint. However, due to welfare concerns, the European Union aims at ending castration of pigs by January 2018 (Declaration of Brussels, 2008), turning boar taint into a challenge in pig production. Although androstenone also contributes to boar taint, the significant contribution of skatole to the meat odor perception indicates that reducing the skatole concentration may be an effective mean to increase consumer acceptance (7, 8). Adding certain dietary fibers (DF) to the feed prevents boar taint by reducing skatole production, making this strategy an alternative to surgical castration (9). Dietary fiber sources rich in inulin, such as chicory roots and Jerusalem artichoke, have been shown as the most effective fiber sources (7, 10–16).
Different DF sources influence the microbial composition and metabolic activity differently. Diets with chicory roots have been reported to result in increased colonic abundance of Megasphaera (M.) elsdenii (17), a dominant butyrate producer in pigs (18, 19). Accordingly, Mølbak et al. (2007) found that feeding a diet with chicory roots and sweet lupins to growing pigs stimulated Bifidobacterium thermoacidophilum and M. elsdenii (20). Further, a Jerusalem artichoke-rich diet resulted in a reduced level of Clostridium (C.) perfringens in both the colon and rectum, and a tendency towards decreased levels of Enterobacteriaceae in the colon (15). Inconsistent effects of inulin on pig gut microbiota have been observed (21). Selective enhancement of beneficial Bifidobacterium and Lactobacillus species was reported after the dietary inclusion of 1.6% (22) and 4% (23, 24) inulin, whereas others did not demonstrate such effect when adding 2% (25) and 3% inulin (26, 27). Butyrate is the major
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fermentation production of inulin (28). Tsukahara et al. (2003) reported increased concentration of short chain fatty acids (SCFA) and the concomitant increased butyrate production when feeding piglets with fructo-oligosaccharides (FOS) (29).
Three hypotheses have been proposed to explain the impact of fermentable DF on skatole production in the hindgut of pigs: 1) supplementation of fermentable DF leads to an increased butyrate concentration in the colon, which reduces cell apoptosis, thereby reducing the availability of TRP for skatole formation from gut mucosa cell debris (30, 31); 2) high levels of fermentable fiber in the hindgut, due to its high levels in the feed, increases the microbial activity, resulting in a higher incorporation of amino acids in bacterial biomass, and a corresponding suppression of proteolytic conversion of TRP into skatole (32, 33); and 3) the effect of DF is elicited by modulating the composition of the microbiota towards one characterized by a decreased abundance of skatole-producing bacteria and an increased abundance of butyrate-producing bacteria.
The experiment outlined in the current study was designed to determine whether, in the absence of high levels of fermentable fibre (chicory roots), butyrate alone elicits a skatole-reducing effect, and whether chicory roots influence the skatole-producing bacteria population in the pig hindgut and thereby decrease the skatole concentration in feces and plasma.
Materials and Methods
Ethics statement
All animal experimental procedures were carried out in accordance with the Danish Ministry of Justice, Law no. 253 of March 2013 concerning experiments with animals and care of experimental animals, and license issued by the Danish Animal Experiments Inspectorate, Ministry of Food, Agriculture and Fisheries, Danish Veterinary and Food Administration.
Animals and experimental design
A total of six crossbred [Duroc × (Danish Landrace × Yorkshire)] entire male pigs with an initial body weight of 41.1 ± 1.8 kg were used. The experiment followed a Latin square design with 3 treatments, 3 periods, and 6 animals, resulting in 2 animals per treatment per period (Figure 1). All animals were
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surgically fitted with a urine catheter into the caecum as described by (34). After one week recovery period in individual pens (1.65 × 1.50 m), the pigs were moved to individual stainless-steel metabolic cages. The duration of each experimental period was 7 days, in which the animals were housed in the metabolic cages and received the experimental treatments. Each experimental period was followed by a two-day period, where the pigs were housed in the same individual pens and fed with a standard Danish grower diet. The weight of feces produced by each pig was recorded daily. On day 6 of each period, pigs were fitted with a permanent jugular vein catheter. Further, on the same day, a fecal sample from each pig was taken directly from the rectum for Real-time PCR, SCFA, dry matter, and phenolic- and indolic compounds determination. On day 7 of each period, blood samples from the jugular vein were taken 30 min before feeding, and at 2, 4, and 6 h after feeding, and analyzed for indolic compound concentration. The collected samples were immediately frozen and stored at -20°C until analyzed.
Diets and infusion
Two diets were formulated: a standard Danish grower diet based on wheat, barley, and soybean meal, and a diet containing 25% chicory root (Table 1). The pigs were fed daily at 07:00 and 14:30. The daily amount of feed offered to the animals was adjusted weekly to 4% of their body weight (BW). One hour after each meal, leftover feed (if any) was removed and recorded. The study included three experimental groups: the Control group received the standard diet and a cecal infusion (25 mL/h) of 0.9% NaCl; the Chicory group received the chicory diet and a cecal infusion (25 mL/h) of 0.9% NaCl; and the Butyrate group received the standard diet and a cecal infusion (25 mL/h) of a 200 mM butyric acid solution, corresponding to a daily infusion of 132 mmol butyrate, constantly during seven days in each period. The amount of butyrate infused was based on the amount estimated to be produced in the hindgut of growers in the study of (35) and on a previous infusion study (36). To obtain a physiological pH of 5.1, 80% of the carboxylic groups of the butyric acid were neutralized by 120 mL/L of a neutralizing solution consisting of 0.13 mol CaCO3/L, 0.65 mol NaCl/L, and 0.50 mol KOH/L (36). To obtain an ionic strength of the butyric acid solution similar to 0.9% NaCl, the butyric acid solution was prepared by dissolving 17 mL butyric acid, 120 mL neutralizing solution, and 500 ml 0.9% NaCl in
363 mL H2O.
Analytical methods
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The concentration of SCFA was determined by gas chromatography as outlined by Canibe and Jensen (2007). The concentration of phenolic- and indolic compounds in feces and indolic compounds in plasma was analyzed by high performance liquid chromatography (HPLC) as described by Knarreborg et al. (2002). The plasma skatole detection level of HPLC was 0.3 ng/mL. The DM content of the fecal samples was measured by freeze-drying.
Microbial genomic DNA extraction
Microbial Genomic DNA (gDNA) from approx. 200 mg feces was extracted using the QIAamp® Fast DNA Stool mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for pathogen detection. The DNA quality was measured by a NanoDrop 1000 spectrophotomer (Thermo Scientific, Waltham, MA), and the DNA concentration in each sample was quantified with the Qubit fluorometer 3.0 (Life Technologies, Grand Island, NY). gDNA was stored at -20°C prior to qPCR and 16S rRNA gene sequencing analysis.
Quantitative real-time PCR
Quantitative real-time PCR for enumeration of O. scatoligenes in feces was performed based on a previously developed Taqman-MGB qPCR assay (Li et al. 2017), using a ViiATM 7 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) associated with the ViiA™ 7 RUO software version 1.2.1 (Life Technologies). The species-specific primer and probe pair used was OscF: 5'-CTTACCAGGGCTTGACATCTTGG-3' (positions: 949-971), OscR: 5'-ACGACACGAGCTGACGACAG-3' (positions: 1043-1024) (obtained from DNA Technology A/S, Denmark), and OscPR: 5'-6-FAM-ACCTGTCTTGGCTCCT-MGB-NFQ-3' (positions: 1014-999) (purchased from Applied Biosystems, Life Technologies, UK). Amplifications were carried out in a total volume of 10 µL consisting of 5.0 µL TaqMan Universal master mix (Applied Biosystems), 300 nM of each primer, and 200 nM of TaqMan-MGB probe and 2 µL of gDNA template. Approximately 10 ng DNA as measured by Qubit® 2.0 fluorometer (Invitrogen) were used. The qPCR program was: hold 2 min at 50 °C, followed by 10 min at 95 °C, then 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Each qPCR run inclided a negative control, a gDNA standard consisting of O. scatoligenes SK9K4T gDNA with known concentrations, and a reference standard consisting of a serial dilution of DNA from 200 mg of feces spiked with 1.5 ×1010 cells/g SK9K4T. All reactions were conducted in triplicate. All
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real-time PCR data were quantified against the reference standard. Results were reported as log O. scatoligenes cells/g feces (wet weight).
16S rRNA gene sequencing
The V3-V4 regions of the 16S rRNA gene were amplified according to the 16S metagenomic sequencing library preparation protocol (Illumina, San Diego, USA) as described by (39) with modifications. Briefly, the first PCR reaction, with the bacterial primers Bac341F: 5'-CCTACGGGNGGCWGCAG-3' (positions 341-357) and bac805R: 5'-GACTACHVGGGTATCTAATCC-3' (positions 785-805) (40) was performed using a 25 µl assay containing 12.5 µL 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems Inc., Wilmington, USA), 0.5
µL forward primer (0.1 µM), 0.5 µL reverse primer (0.1 µM), 1.0 µL BSA, 8.5 µL H2O, and 2.0 µL DNA template. The program was: 95 °C for 0.5 min, and 20 cycles of 55 °C for 0.5 min, 72 °C for 0.5 min, and 72 °C for 5 min. The second PCR reaction, amplified with the overhang adapters (5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-(locus-specific sequence)-3´ and 5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-(locus-specific sequence)-3´), was performed following the same procedures except that only ten amplification cycles were conducted. The third PCR amplification, with added Nextera index primers (Illumina Nextera XT v2, Illumina, San Diego, USA), was performed following the same procedures except that only eight amplification cycles were conducted. The PCR products from each reaction were purified using magnetic bead technology (Agencourt AMPure XP, Beckman Coulter Inc., Brea, USA) according to the manufacturer´s instructions. The size and the quality of all amplicons were checked using 1% agarose gel electrophoresis. The final DNA concentration of all libraries was quantified fluorometrically by Qubit using the high-sensitivity assay kit and then pooled in equimolar concentrations and diluted to a final DNA concentration of 4 nM. The sample pool was sequenced with an Illumina MiSeq (Microarray Facility, VUmc, Amsterdam, NL) following a 2 × 300 bp paired-end protocol at the Center for Geomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmark.
Data availability
The raw reads used in this study were deposited to the Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRP089699.
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Bioinformatic analysis
Raw Illumina FASTQ sequences were initially demultiplexed using Miseq Reprotrer V2.0. and then analyzed using QIIME (Quantitative Insights Into Microbial Ecology) software package version 1.9.1 (41). Paired-end reads were first merged by multiple_join_paired_ends.py and followed by extract_barcodes.py to remove forward and reverse primer sequences. In QIIME, multiple_split_libraries_fastq.py was used to demultiplex and quality filter, using a minimum quality score of 25. The pick_open_reference_otus.py script (42) in QIIME was used to pick OTUs and assign taxonomy using the uclust method (43) with Greengenes references (13_8 release) database (44), and a 97% similarity threshold. For Olsenella taxonomic classification, the final sequences generated from QIIME split_libraries were analyzed using the Ribosomal Database Project (RDP) classifier (https://pyro.cme.msu.edu/classifier/form.spr) (45) with a 80% confidence threshold. OTUs representing fewer than 0.005% of all sequences were discarded using filter_otus_from_otu_table.py script (46). Alpha- (estimated by Phylogenetic Diversity (PD) Whole Tree, Chao 1 and Observed Species indices) and beta diversity (estimated through UniFrac distances) were conducted using the QIIME workflow core_diversity_analysis.py, with a sampling depth of 39,346 (47). R version 3.2.5 (R Development Core Team 2014) with the R package ampvis v.1.25.0 (https://github.com/MadsAlbertsen/ampvis) (48) was further used for the analysis of the sequencing data. Principal component analysis (PCA) was conducted, using square root transformed OTU counts in Vegan (http://CRAN.R-project.org/package=vegan). OTUs in differential abundance between treatments were identified by DESeq2 (49) using test = “wald” and fitType = “parametric”.
Statistical analysis
The model used to analyze the variables from feces was a mixed model including treatment, period, and their interaction as fixed effects. The repeated statement was used to account for correlation among samples taken from the same pig. Compound symmetry was used as the covariance structure. The model used to analyze the plasma data included treatment, period, time of sampling, and their interactions. The repeated statement was included to account for the various samples taken from the same pig at various time points, and the random pig effect to account for samples taken from the same pig in the three periods. Autoregressive was used as the covariance structure. The statistical analyses were performed with SAS (version 9.4, SAS Inst. Inc., Cary, NC). Differences between means were
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compared by Tukey’s least significant difference. Significance was declared at p$<$0.05.
Results
The initial BW of the animals was 41.1 ± 1.8 kg, and at the end of the experimental period, the BW was 62.4 ± 4.5 kg. Feed intake in the Control, Chicory, and Butyrate groups was 1.77, 1.80, and 1.68 kg/d, respectively. During the whole study period, the pigs gained 0.86 (Control), 0.85 (Chicory) and 0.78 kg/d (Butyrate). During the first period, one pig in the Butyrate group lost 0.9 kg weight. During the third period, one pig in the Control and one in the Chicory group had diarrhea and received antibiotic (Streptipenprokain Rosco Vet) treatment for three days.
Amount of feces excreted and fecal SCFA concentration
A significant effect of the interaction between treatment and period was observed for ratio of feces to feed intake, and feces DM (Table 2). Evaluating the data, this could be ascribed to a low value of the ratio in the Control period 3 and a high value of DM in the Chicory in period 2. No impact of treatment was found on the fecal SCFA concentration except branched chain fatty acids (BCFA) (Table 2). A significant effect of the interaction between treatment and period was detected, probably due to a high level of BCFA concentration in the Chicory in period 2. However, the overall tendency was that the BCFA (isobutyric and isovaleric acid) concentration was numerically higher in the Butyrate group than in the other two groups (ptreatment = 0.10).
Indolic- and phenolic compounds in feces
No significant impact of treatment was found on the fecal content of tryptophan, indole propionic acid
(IPA) and Indole acetic acid (IAA), but a tendency for IPA (ptreatment = 0.10) was observed, with the highest value measured in the Chicory group (Table 2). A decrease in fecal skatole concentration was found in animals receiving chicory roots, being significantly different from the Butyrate group (3.8 vs
40.8 mmol/kg, ptreatment = 0.03). A significant effect of the interaction between treatment and period was observed for indole and p-cresol, which could be explained by a high level of indole and p-cresol concentration in the Chicory group in period 2. However, the average fecal p-cresol concentration was numerically highest in the Butyrate group (ptreatment = 0.05).
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Skatole and indole concentration in plasma
Sampling time (from 30 min before feeding to 6 h after feeding) had no effect on the levels of plasma skatole or indole (data not shown). In accordance with the fecal data presented in Table 2, average plasma skatole levels for the three periods were numerically lowest (0 ng/mL) in the Chicory group and highest (2.0 ng/mL) in the Butyrate group (Figure 2A). However, due to high variation among pigs and a significant effect of period, a significant difference among treatments was only observed in the first period, where a higher concentration of skatole in the Butyrate group was detected compared to the Chicory group (4.7 vs 0 ng/mL, p = 0.01), with the same tendency when compared to the Control group (4.7 vs 1.7 ng/mL, p = 0.10) (Figure 2A). The plasma indole concentrations at the tested time points were not affected by either chicory roots supplementation or butyrate infusion (Figure 2B).
Olsenella scatoligenes and Olsenella abundance in feces
Although the lowest numerical fecal and plasma skatole concentrations were found in the Chicory group, an increase of O. scatoligenes numbers was detected in this group compared to the Control group (6.2 vs 4.7 log10 cells/g, p = 0.06) (Figure 3A). Similar to O. scatoligenes, Olsenella abundance in feces was higher in the Chicory group compared to the Control and Butyrate groups (0.52% vs 0.06% and 0.05%, respectively), but no significant difference was achieved (Figure 3B).
The overall fecal microbiota of entire male pigs
A total of 18 fecal samples were collected in the current study, and a total of 1,200,679 high-quality sequences with a minimum of 40,833 sequences per sample were obtained. After removal of low-abundant OTUs (< 0.005%), the data contained 994 OTUs, representing 17 phyla and 95 genera. The microbiota across all samples was dominated by the Firmicutes and Bacteroidetes phyla, accounting for 75.7% of total sequences (Figure 4A), and the average ratio between Firmicutes and Bacteroidetes for all treatments was between 1.9 and 2.5. Other phyla were present at percentages lower than 10% (e.g. Spirochaetes (7.7%), Proteobacteria (7.6%), Lentisphaerae (2.3%), Cyanobacteria (1.9%), and Tenericutes (1.0%)). In general, the abundance of the various phyla in the different treatments was rather similar.
The classified bacterial genera detected at ≥ 1% average relative abundance were (in decreasing order):
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Treponema, RFN20, Oscillospira, Prevotella, Campylobacter, Succinivibrio, CF231, Lactobacillus, Anaerovibrio, Mistuokella and Catenibacterium (Figure 4B). In addition, the genera Roseburia, Dorea, Bacteroides, Coprococcus, and Ruminococcus also accounted for ≥ 1% of the relative abundance in the control pigs (data not shown).
Changes in the relative abundance of genera among the different treatments were compared by DESeq wald test. A total of 6 genera (Bacteroides, Collinsella, and 4 unclassified) had significantly different abundances (p < 0.05), and 10 genera (Roseburia, Paludibacter, Enterococcus, Coprococcus, and 6 unclassified) tended (p ≤ 0.10) to be different among the different treatments (Table 3). Bacteroides, Roseburia, Enterococcus, and Coprococcus were most abundant in the Control group; Collinsella most abundant in the Chicory group, while Paludibacter was most abundant in the Butyrate group. Six of the unclassified genera belonged to the Firmicutes, two belonged to Bacteriodetes, and one each belonged to Proteobacteria, and Cyanobacteria, respectively.
Alpha diversity
Samples were rarefied to 39,346 sequences to account for unequal numbers of sequences between samples before calculating α-diversity indices (Figure 5). The average of Chao 1 (a non-parametric estimator of OTU richness) in the Control, Chicory, and Butyrate group was 676, 566, and 715, respectively; the average number of observed OTUs (a pure estimator of community richness) was 605, 502, and 650 respectively; and the average value for Phylogenetic Distance (PD) (the degree of phylogenetic divergence between sequences within each sample) was 46.3, 41.4, and 49.6 respectively. Overall, all alpha diversity indices revealed that there were significant differences in community richness between the Butyrate and Chicory groups (p < 0.05), whereas the Control group had intermediate values. A similar pattern among the different treatments was further confirmed by cumulative rank abundance curves (Figure 5).
Beta diversity
Principal component analysis (PCA) and principal coordinate analysis (PCoA) were used to estimate β-diversity and to compare the three treatments (Figure 6). The observed community composition of the Chicory group was distinguished from the Butyrate and Control groups, whereas there was an overlap between the Control and the Butyrate groups (Figure 6A). Analysis of similarities (ANOSIM)
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of unweighted UniFrac distances indicated that the Control, Chicory, and Butyrate groups were significantly different (p = 0.010, R = 0.260). The PCoA plot of the unweighted UniFrac distances visually confirmed that the Chicory group formed a distinct cluster separated from the Butyrate and the Control groups (Figure 6B). In contrast, no clear visual separation among the treatments was observed in the PCoA plot of the weighted UniFrac distances (Supplementary Figure 1), despite the ANOSIM of weighted UniFra also showed that the treatments were significantly different (p = 0.040, R = 0.162).
Discussion
The origin of the TRP as substrate for microbial production of skatole can be from either dietary or endogenous protein sources (50, 51). However, addition of free TRP above the requirements to pig diets had no effect on skatole levels (52), most probably because free TRP is absorbed in the small intestine, and therefore not available for microbial fermentation in the hindgut. Thus, cell debris resulting from apoptosis of intestinal mucosa cells has been proposed to be a major substrate for skatole formation (50, 51). According to Claus et al. (2003), the reduced skatole levels observed when feeding raw potato starch to male pigs is due to inhibition of colonocyte apoptosis by the butyrate produced by microbial fermenting the raw potato starch (31). A lower content of cell debris in the lumen leads to a lower availability of endogenous TRP from cell debris for skatole production. As a consequence, less skatole is produced in the pig colon (31, 53). However, addition of inulin-coated butyrate to the feed, in which the butyrate is expected to be released in the hindgut, has not shown to have an effect on skatole levels in colon, plasma, or adipose tissue (54).
We found that a physiologically relevant rate of intracecal butyrate infusion, which did not cause obvious health problems to the pigs, did not decrease skatole levels in feces or plasma. In fact, higher values of skatole were detected, with significant differences detected when compared to the Chicory group. Moreover, intracecal butyrate infusion did not result in an increased concentration of SCFA (including butyrate) in feces. In general, a tendency for increased levels of p-cresol, and BCFA was measured in the feces of pigs receiving intracecal butyrate infusion. p-Cresol, isobutyrate, and isovalerate are formed from tyrosine (55), valine, and leucine (56) degradation, respectively. These findings indicate that protein fermentation was somewhat increased in response to intracecal butyrate infusion. This could be a result of a stimulation of certain proteolytic bacteria in response to exogenous butyrate. Although changes in fecal microbiota composition in Butyrate group also indicate such effect,
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the data is not solid enough to make clear conclusions on this aspect.
The higher measured level of skatole in the animals receiving exogenous butyrate was unexpected. One explanation could be increased cell apoptosis either in the small intestine or the large intestine due to the butyrate infusion. It is worth noting that the effect of cecal butyrate infusion on intestinal cell proliferation and apoptosis is controversial (57, 58). Further, the in vivo effect of butyrate on cell apoptosis and proliferation has been found to be dependent on the dietary lipid source (59). Whether butyrate stimulates (proliferation) or inhibits (apoptosis) cell growth may depend on the availability of other specific energy sources, providing an explanation for the apparent contradictory effects of butyrate on cell growth. The current results indicate that exogenous butyrate alone does not prevent skatole formation; in fact, a tendency towards the opposite was observed.
Butyrogenic and bifidogenic effects of inulin-type fructans have been well established in humans in various studies (60–62). However, in the present study, fecal SCFA levels, including butyrate, were not affected by the high level of dietary chicory roots supplementation. In line with our study, Rideout et al. (2004) and Vhile et al. (2012) found no impact of feeding diets containing 5% inulin extract, 9% chicory inulin, or 12.2% Jerusalem artichoke on fecal butyrate concentration (10, 15). Similarly, in humans, no significant changes were observed in fecal SCFA levels following inulin supplementation (63, 64). Since up to 99% of the SCFA produced in the hindgut are absorbed rapidly (65), butyrate levels in feces do not accurately reflect its production or concentration in the hindgut. This was also pointed out by Vhile et al. (2012) (15).
Microbial degradation of TRP results in the production of skatole, indole and IPA, with IAA as an intermediate product in skatole production (38). In our study, feeding high levels of chicory roots showed no effect on fecal indole and IAA concentrations, a reduction in fecal skatole concentration, and a tendency towards an increased level of IPA. Similar results were found by Knarreborg et al., (2002) when feeding entire male pigs with a diet supplemented with 10% sugar beet pulp. These data may indicate that DF fermentation altered the microbial metabolism of TRP towards IPA production at the expense of skatole. However, due to the typically low concentration of IPA measured, IPA is not considered to totally account for the reducing effect of DF on skatole production. It has been suggested from in vitro studies that the skatole-reducing effect of sugar beet pulp and FOS is through shifting the microbial metabolism of TRP to indole production at the expense of skatole (33, 66). Vhile et al.
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(2012) proposed a similar mechanism, although found, as in the current study, that the decreased skatole production was not accompanied with an increased indole levels.
Until now, bacteria confirmed to produce skatole belong to only two genera, i.e., Clostridium (67) and Olsenella (3). Unlike C. scatologenes and C. drakei, which have been isolated from soil and sediment, respectively, O. scatologenes is the only reported skatole producer isolated from the pig gut (3). Adding Jerusalem artichoke to feed reduced the growth of C. perfringens, which the authors postulated could potentially be used as an indicator of the Clostridium genus in general. Thus, they hypothesized that adding high levels of dietary fermentable fiber results in reduced growth of the skatole-producing bacteria Clostridium in the hindgut of pigs, and hence reduced skatole synthesis (15). However, in the current study, no inhibitory effect of chicory roots on Clostridium was observed. Further, in our study, the skatole-reducing effect of chicory roots did not coincide with a reduced number of the skatole producer O. scatologenes. On the contrary, an increased number of O. scatologenes by chicory roots was detected, which is in line with the studies of (68, 69), who reported increased proliferation of Olsenella genus by DF.
One possible explanation for the observed proliferation of O. scatoligenes in the Chicory group could be that skatole production, which is not an essential metabolic function of O. scatoligenes, is suspended during the exponential growth phase when there is sufficient supply of fermentable carbohydrates. At the same time, available amino acids, including TRP, are incorporated into bacterial biomass instead of being used for skatole production, leading to bacteria growth. On the other hand, in the Control and the Butyrate groups, with less available carbohydrates, O. scatoligenes starts to draw its energy for maintenance from protein fermentation resulting in a higher production of skatole as an end product, while bacteria growth is hindered. Thus, it can be implied that the reducing effect of chicory roots on skatole production is not by inhibiting this skatole-producing bacteria growth in the pig hindgut, but rather by modulating the activities of the gut microbiota.
While the reported relative abundance of various bacteria phyla in pig gut is somewhat variable in the literature, Firmicutes and Bacteroidetes are consistently the two most dominant phyla measured (70–73). In line with this, the two most abundant phyla in our study, representing approximately 76% of the total sequences regardless of treatment, were Firmicutes and Bacteroidetes regardless of treatment. Cyanobacteria, although being photosynthetic organisms, were evident in pig feces. The
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numerically higher recovered Cyanobacteria in the Chicory group might be of feed origin. Surprisingly, in the present study, Treponema, accounting for approximately 90% of Spirochaetes, was the most abundant genus, which is in contrast to previous reports finding Prevotella as the dominant genus in the pig gut (73–75). The growth of Treponema spp. can be enhanced by carbohydrates (76). Niu et al. (2015) also found Treponema as one of the most abundant genera and its abundance being positively correlated with apparent crude fiber and acid detergent fiber digestibility (71). In addition, Anaerobacter, Prevotella, Lactobacillus, Oscillospira, and Roseburia are common abundant genera (> 1% of the total sequences) found in pig gut (73, 77). Differences in animal genetics, ages, diet formulations, environments, as well as sequencing methods may result in discrepancies among studies, and thereby direct comparison of metagemomic data across studies can be difficult.
There were no significant differences among treatments in the proportion of taxonomic groups of the fecal microbiota at the phylum level. However, there were statistical differences when measured at the genus level, yet only limited to a few genera. The abundance of Bacteroides, Collinsella, Roseburia, Paludibacter, Enterococcus, and Coprococcus were significantly influenced by treatment. Cho et al. (2015) found that the production of skatole and p-cresol is positively correlated with Bacteroides (78), whereas no such relationship was observed in our study. Chicory roots and butyrate inclusion resulted in a decreased abundance of Bacteroides, However, chicory roots and butyrate had an opposite effect on skatole formation. We observed that the Butyrate group contained the highest proportion of unclassified genera within Clostridiales (1.3%) and Bacteroidales (3.6%), which contain members of proteolytic species that are capable of metabolizing aromatic amino acids (e.g. tryptophan and tyrosine) (79). It may be speculated that the enrichment of these proteolytic species was responsible for the observed skatole and p-cresol increase after butyrate infusion. A more thorough analysis at species level would however be needed in order to prove this statement.
With regard to the butyrate-producing bacteria, Acidaminococcus and Megasphaera within clostridial clusters IX, and Coprococcus, Eubacterium, and Roseburia within clostridial clusters XIVa, are dominant in the pig gut microbiota (19). M. elsdenii is a well-known lactate fermenter (to butyrate and propionate), and its abundance has been found to increase when pigs are fed a diet added chicory roots (17, 20). In our study, Megasphaera was numerically higher, while Roseburia was significantly lower when chicory roots were added to the diet. Therefore, chicory roots may selectively promote the growth of specific butyrate-producing bacteria such as M. elsdenii in pig hindgut. However, the skatole
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reducing effect of chicory roots was not associated with a general increase in the population of butyrogenic bacteria.
Alpha-diversity indices and rarefaction analysis showed lower bacterial species richness in the fecal microbiota of pigs receiving chicory roots than in those receiving butyrate infusion. Similarly, Heinritz et al. (2016) found lower total bacterial numbers in cecal digesta of pigs fed a low-fat/high-fiber diet (80). Varel et al. (1982) showed an initial decrease in total bacterial counts in rectal samples when pigs fed with high-fiber diet. The total counts, however, increased after continuous fiber-feeding for 12 weeks, suggesting that the microbiota is initially suppressed when exposed to a high-fiber diet, followed by and adaption process and a reestablishment of itself.. Beta diversity analysis revealed an influence of treatment on fecal microbiota composition. Both PCA and unweighted UniFrac PCoA plots showed that the Chicory group formed a distinct cluster, whereas the Control and Butyrate groups, which were both fed a standard Danish diet, clustered more closely. However, a clear separation was not observed in the weighted UniFrac PCoA plots. Weighted UniFrac (quantitative) accounts for the relative abundance of OTUs, whereas unweighted UniFrac (qualitative) only considers their presence or absence (47). Unweighted unifrac is most informative when communities differ primarily by their membership, in part because abundance information can obscure significant patterns of variation in which taxa are present (82). The current data indicated that chicory root supplementation had an effect on the microbiota by affecting the presence or absence of OTUs rather than modulating their relative abundances.
Conclusion
Intracecal infusion of butyrate did not decrease the concentration of skatole in faeces or plasma, what indicates that the skatole-reducing effect of chicory roots is not through increased butyrate production in the hindgut. Further, addition of chicory roots to the diet did not result in a reduced abundance of the skatole-producing bacterium O. scatoligenes, which suggests that the impact of chicory roots on skatole is not via inhibition the growth of skatole-producing bacteria. The most likely mechanism behind the reducing effect of chicory roots on skatole formation is through incorporation of more amino acids (including TRP) into bacterial biomass due to its high fermentability, and a corresponding suppression of proteolytic conversion of TRP into skatole. However, further evaluation of microbial activities would be required to confirm the effect of DF on the gut microbiota and the formation of
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skatole in the hindgut of entire male pigs.
Acknowledgments
This work was supported by the Danish AgriFish Agency, Ministry of Food, Agriculture and Fisheries (Project 3405-10-OP-00134). L.X. was supported by a scholarship from the China Scholarship Council (CSC). We want to thank Trine Poulsen and Thomas Rebsdorf for skilful technical assistance, and Samantha Joan Noel and Ann-Sofie Riis Poulsen for bioinformatic analysis assistance. We also appreciate help from Henry Johannes Høgh Jørgensen for assisting to perform the catheter surgery.
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74. Lamendella R, Domingo JWS, Ghosh S, Martinson J, Oerther DB. 2011. Comparative fecal metagenomics unveils unique functional capacity of the swine gut. BMC Microbiol 11:103.
75. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Moøller K. 2002. Culture-independent analysis of gut bacteria: The pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 68:673–690.
76. Nordhoff M, Taras D, Macha M, Tedin K, Busse HJ, Wieler LH. 2005. Treponema berlinense sp. nov. and Treponema porcinum sp. nov., novel spirochaetes isolated from porcine faeces. Int J Syst Evol Microbiol 55:1675–1680.
77. Kim HB, Borewicz K, White B a., Singer RS, Sreevatsan S, Tu ZJ, Isaacson RE. 2012. Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter, tylosin. Proc Natl Acad Sci 109:15485–15490.
78. Cho S, Hwang O, Park S. 2015. Effect of dietary protein levels on composition of odorous compounds and bacterial ecology in pig manure. Asian-Australas J Anim Sci 28:1362-70.
79. Russell WR, Duncan SH, Scobbie L, Duncan G, Cantlay L, Calder AG, Anderson SE, Flint HJ.
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80. Heinritz SN, Weiss E, Eklund M, Aumiller T, Heyer CME, Messner S, Rings A, Louis S, Bischoff SC, Mosenthin R. 2016. Impact of a high-fat or high-fiber diet on intestinal microbiota and metabolic markers in a pig model. Nutrients 8:E317
81. Varel VH, Pond WG, Pekas JC, Yen JT. 1982. Influence of high-fiber diet on bacterial populations in gastrointestinal tracts of obese- and lean-genotype pigs. Appl Environ Microbiol 44:107–112.
82. Lozupone CA, Hamady M, Kelley ST, Knight R. 2007. Quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities. Appl Environ Microbiol 73:1576–85.
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Table 1 Ingredients and the calculated composition of the experimental diets.
Item Control/Butyrate Chicory Ingredient composition (%) Wheat 55.97 28.84 Barley 20.00 20.00 Dehulled toasted soybean meal 9.93 12.00 Dehulled sunflower cake 8.00 8.00 Dried milled chicory roota - 25.00 Soybean oil 1.70 1.40 Sugar beet molasses 1.50 2.00 Calcium carbonate 1.42 1.33 Monocalcium phosphate 0.36 0.35 Sodium chloride 0.49 0.48 L-Lysine-HCl, 98% 0.32 0.29 DL-Methionine , 98% 0.03 0.04 Threonine, 98% 0.08 0.08 Vitamin and mineral premixb 0.20 0.20 Calculated composition (g/kg DM)
DM 862 886 Fat 37 30 Crude protein 151 150 Ash 50 59 Calcium 7.2 8.4 Phosphorus 4.5 4.5 a The content of fructan in the dried milled chicory root was 65.2 %. b Supplied per kilogram of diet: 4200 IU of vitamin A; 420 IU of vitamin D3; 69.2 mg of vitamin E; 2.1 mg of vitamin B1;
2.1 mg of vitamin B2; 3.2 mg of vitamin B6; 20 µg of vitamin B12; 63.0 mg of DL-alpha-tocopherol; 69.2 mg of
DL-alpha-tocopherol acetate; 10.5 mg of Ca-D-pantothenic acid; 21 mg of niacin; 20 µg of biotin; 2.1 mg of vitamin K3; 84 mg of Fe (Fe (II) sulphate); 15 mg of Cu (Cu(II) sulphate) and 42 mg of Mn (Mn(II) oxide).
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Table 2 Impact of the experimental treatments on the feces/feed ratio, DM, concentration of organic acids and chemical compounds in feces.
Treatment S.D. P-value Control Chicory Butyrate Treatment1 Period Treatment × period Feces/feed (g/kg) 478.3 605.7 513.7 47.3 0.31 0.38 0.03 Feces DM (%) 22.6 20.4 24.6 1.1 0.12 0.28 0.03 Organice acid (mmol/kg of wet feces) Acetic acid 75.9 79.3 74.3 4.4 0.62 0.86 0.48 Propionic acid 33.9 37.2 33.4 3.5 0.57 0.98 0.96 Butyric acid 21.8 16.2 17.9 4.2 0.55 0.93 0.79 Valeric acid 4.9 5.9 5.9 1.2 0.84 0.71 0.98 BCFA2 4.9 4.7 7.8 0.8 0.10 0.31 0.02 Chemical compounds (mg/kg of wet feces) Tryptophan 7.4 5.9 1.5 3.3 0.32 0.30 0.89 Indole propionic acid (IPA) 1.8 6.3 0.8 1.6 0.10 0.49 0.57 Indole acetic acid (IAA) 3.0 1.0 0.4 1.3 0.44 0.61 0.71 Skatole 15.7ab 3.8a 40.8b 6.5 0.03 0.29 0.32 Indole 16.8 17.1 15.8 4.7 0.98 0.39 0.09 p-cresol 58.0 63.1 118.9 12.9 0.05 0.28 0.02
1 Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl.
2BCFA (branched chain fatty acids): including isobutyric and isovaleric acids ab Different letters indicate significant differences (P < 0.05).
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Table 3 Impact of the experimental treatments on the relative abundances (%) of bacterial taxa groups in the fecal microbiota of pigs.
Taxonomy1 Treatment2 P-value Control Chicory Butyrate Proteobacteria; o_GMD14H09_New.ReferenceOTU57 2.20 - - < 0.01 Bacteroidetes; Bacteroides 1.45 - - 0.01 Firmicutes; f_Ruminococcaceae_344523 0.11 0.02 - 0.02 Firmicutes; o_Clostridiales_187767 - - 1.31 0.02 Cyanobacteria; o_YS2_New.ReferenceOTU1001 - 0.11 0.19 0.02 Actinobacteria; Collinsella 0.25 0.41 0.01 0.03 Bacteroidetes; f_S24-7_ New.ReferenceOTU3921 0.40 - - 0.05 Firmicutes; f_Lachnospiraceae_708680 1.77 0.31 0.16 0.05 Firmicutes; Roseburia 1.93 0.18 0.14 0.05 Firmicutes; o_Clostridiales_New.ReferenceOTU3458 - - 0.41 0.06 Firmicutes; f_Lachnospiraceae_183362 - - 0.11 0.07 Bacteroidetes; Paludibacter - - 0.09 0.08 Firmicutes; Enterococcus 0.16 - - 0.08 Firmicutes; Coprococcus 0.87 0.61 0.18 0.10 Firmicutes; f_Lachnospiraceae_ New.ReferenceOTU522 0.23 - 0.03 0.10 Bacteroidetes; o_Bacteroidales_ New.ReferenceOTU4486 0.06 - 3.60 0.10
1Taxonomy shown as a phylum and genus classification where they could be classified to a genus, otherwise they are shown at phylum and OTU classification. Only genera significantly affected (p ≤ 0.05) or with tendency to be affected (p ≤ 0.10) by treatment are presented. -, indicates trace abundance (< 0.01%).
2 Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl.
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FIG 1 Distribution of pigs in diets and periods. Six animals were surgically fitted with a urine catheter in the cecum. Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl. The duration of each experimental period was 7 days, in which pigs were housed in metabolic cages, followed by a two-day period where pigs were moved to individual pens and fed a standard Danish diet.
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FIG 2 Plasma (A) skatole and (B) indole concentrations in the pigs from three experimental treatments and in the three experimental periods. Within each period, blood samples were taken from two pigs in each group at -30 min, 2, 4, and 6 h. Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl. Data are presented as means. ab Different letters indicate significant differences (P < 0.05).
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FIG 3 Fecal (A) O. scatoligenes number, determined by quantitative real-time PCR and presented as log10 cell/g wet feces; and (B) Olsenella abundance, classified by RDP classifier and presented as read abundance % in the pigs from three experimental treatments. Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl.
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FIG 4 Overall fecal microbiota composition: (A) Relative abundances of the 10 most abundant phyla in the three treatments; (B) Relative abundances of the 20 most abundant genera in the three treatments. The taxonomy is shown as the phylum and genus classification where they could be classified to a genus, otherwise they are shown as the phylum and OTU classification. Control: pigs received a standard diet and a cecal infusion of NaCl; Butyrate: pigs received a standard diet and a cecal infusion of butyrate; Chicory: pigs received a chicory diet and a cecal infusion of NaCl.
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FIG 5 Alpha-diversity of fecal samples from the three experimental treatments, using cumulative rank abundance curves and diversity indices (Chao 1, observed OTUs and PD whole tree). Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl. ab Different letters indicate significant differences (P < 0.05).
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FIG 6 Beta-diversity: (A) Principle component analysis (PCA) based on the square root transformed OTU counts; (B) Principal coordinate analysis (PCoA) based on unweighted Unifrac metrics of the fecal samples from the three experimental treatments (each point represents the composition of fecal microbiota of one pig). Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl.
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Supplementary FIG 1 Principal coordinate analysis (PCoA) based on weighted Unifrac metrics of fecal samples from the three experimental treatments (each point represents the composition of fecal microbiota of one pig). Control: pigs received a standard diet and cecal infusion of NaCl; Butyrate: pigs received a standard diet and cecal infusion of butyrate; Chicory: pigs received a chicory diet and cecal infusion of NaCl.
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11. General discussion
Adding certain fermentable DF is one of the feasible alternatives to reduce skatole production in the pig hindgut. The most effective DF seems to be inulin rich feed components such as chicory root (Jensen et al. 2014). The exact mechanism underlying the skatole reducing effect of chicory roots remains unclear, but three main hypotheses have been put forward (Figure 7): 1) supplementation of fermentable DF leads to an increased butyrate concentration in the colon, which reduces cell apoptosis, thereby reducing the availability of TRP for skatole formation from gut mucosa cell debris (Claus et al. 1994; Claus et al. 2003); 2) high levels of fermentable fiber in the hindgut, due to its high levels in the feed, increases the microbial activity, resulting in a higher incorporation of amino acids in bacterial biomass, and a corresponding suppression of proteolytic conversion of TRP into skatole (Jensen et al. 1995a; Xu et al. 2002); and 3) DF fermentation alters gut microbiota composition by decreasing skatole-producing bacteria and increasing butyrate-producing bacteria.
Figure 7 Potential mechanisms of skatole-reducing effect of dietary fiber.
In order to test these hypotheses, four main studies were carried out with the aim of i) screening and characterizing skatole-producing bacteria from pig hindgut (paper I, II and III); ii) establishing a Taqman-MGB real-time PCR assay to quantify skatole-producing bacteria from pig hindgut (paper 123
IV); iii) exploring phylogenetic diversity of butyrate-producing bacteria from pig hindgut (paper V); and iv) establishing the mode of action behind the reducing effect of DF (chicory roots) on skatole production in the entire male pig hindgut (paper VI).
11.1. Skatole-producing bacteria
Production of skatole involves a two-step process in which the precursor TRP is first deaminated to indole acetic acid, which is then further decarboxylated to skatole (Jensen et al. 1995b). While a variety of bacteria metabolize TRP to indole and IAA, few bacteria have been reported to catalyze the step from IAA to skatole (Deslandes et al. 2001), and only two of these, Clostridium scatologenes (isolated from soil) and Clostridium drakei (isolated from sediment), have been fully characterized and made available in culture collections (Whitehead et al. 2008). Yokoyama et al. (1977) isolated a Lactobacillus strain from bovine rumen that produced skatole from IAA. More recently, Attwood et al. (2006) reported six rumen organisms (similar to Prevotella sp., Clostridium sp., Actinomyces sp. and Megasphaera sp.) that produced skatole in the presence of IAA, suggesting that the microbial population of skatole-producers in the rumen is more diverse than previously assumed. However, to our knowledge, these isolates have not been further characterized or made available in culture collections. Further, so far, no skatole-producing bacterium has been isolated from the pig gut. Isolation and characterization of such an organism would provide insight into skatole production and regulation, hence potentially facilitating the reduction of boar taint in pigs.
In the present project, we screened 122 OTUs, which represent 2678 isolates from the GI-tract of pigs for their ability to produce skatole. The results showed that, except O. scatoligenes SK9K4 (Paper I and II), none of these isolates produced skatole from TRP or IAA. Simultaneously, we tested other members of the Olsenella genus. O. uli (isolated from either human gingival crevices or periodontal pockets), but not O. profusa or O. umbonata, was able to produce skatole. However, unlike C. scatalogenes and C. drakei, which produce skatole directly from TRP (Whitehead et al. 2008), O. scatoligenes and O. uli synthesize skatole by decarboxylating IAA, but are unable to produce skatole directly from TRP (paper I). Thus, the genome sequence of O. scatoligenes (reported in paper II), is of interest as it could potentially be used to elucidate the skatole metabolic pathways, hence facilitating the reduction of skatole production in pigs.
The 2.47 Mbp draft genome sequence of O. scatoligenes SK9K4T contains 4-hydroxyphenylacetate decarboxylase (4-Hpd) (EC 4.1.1.83) genes, verifying that it possesses the tyrosine degradation IV
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(to p-cresol) pathway (paper II). Moreover, O. scatoligenes also has an aliphatic amidase (EC 3.5.1.4) gene, which is required to produce IAA from tryptophan (Li et al. 2016b). However, O. scatoligenes is not able to produce IAA. Since the enzymology of skatole synthesis has not been characterized so far, very little is known about the genetics of this pathway. Nevertheless, candidate genes for skatole production can potentially be identified by comparative genomics.
Among the 122 OTUs, we screened a Megasphaera sp. strain DJF_B143 (EU728714), which according to the 16S rRNA gene sequence analysis (paper III), has 99.9% identity with Megasphaera sp. TrE9262 (DQ278866), a bacteria isolated from the rumen of sheep reported to produce skatole (Attwood et al., 2006). However, we observed no skatole production by DJF_B143 either from TRP or IAA when incubated in modified peptone-yeast-glucose or in colon fluid-glucose-cellobiose-agar media. We also sequenced strain DJF_B143 to explore the genomic basis to confirm this observation. The 2.58 Mbp draft genome sequence of Megasphaera sp. DJF_B143 lacks an aliphatic amidase gene, suggesting that this strain is unable to metabolize tryptophan to skatole via IAA (Li et al. 2016c) (paper III), which confirms our results from the incubation studies.
After screening the 122 OTUs, which was considered to represent the gut microbiota of pigs, O. scatoligenes was the only skatole-producing bacterium, and is the only known skatole-producing bacterium isolated from pig GI-tract so far (Li et al. 2015). Therefore, these results indicate that skatole-production is limited to a few species, and O. scatoligenes seems to be the main dominant skatole-producing bacterium in the pig GI-tract.
11.2. Quantification of skatole-producing bacteria
After identification of the skatole-producing bacteria in the pig hindgut, quantification of their presence can be an important mean in the search for strategies to reduce boar taint via manipulation of these bacteria. As revealed by 16s rRNA sequencing studies, Olsenella genus constitutes ~ 0.12% (from 0.02% to 0.94%) of the total microbiota in pig feces (Kim et al. 2011). By using the Pig Intestinal Tract Chip, Haenen et al. (2013) reported around 0.01% of Olsenella in the pig cecal content. Yokoyama et al. (1977) isolated a skatole-producing bacterium Lactobacillus sp. strain 11201 that morphologically and physiologically seems to be very similar to O. scatoligenes SK9K4T. But they failed to isolate it directly from serially diluted rumen fluid, indicating that it is not among the predominant rumen bacteria. Further, using the most probable number technique in combination with skatole measurements, it has been estimated that the population of
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skatole-producing bacteria accounts for less than 0.01% of the total microbiota in pig fecal samples (Jensen and Jensen 1998).
A variety of methods are available to quantify gut microbiota. Traditionally, cultivation is the classical approach for the identification and quantification of bacteria. However, it is labor intensive, and only part of gut bacteria is covered. Due to the low abundance of Olsenella presence in the pig gut and lack of an appropriate selective medium for O. scatoligenes so far, it is thus not possible to quantity this bacterium by culture methods. The 16S rRNA gene is a suitable marker gene for taxonomic and phylogenetic quantification of gut microbiota. Examples of the techniques based on 16S rRNA amplicon are qPCR, DGGE, T-RFLP, FISH, DNA microarrays, and HTS technologies (Fraher et al. 2012). Among them, DGGE, T-RFLP, FISH, and DNA microarrays are semi-quantitative. One shortcoming of current HTS methods is the limitation of the methods to resolve bacteria at or below the genus level (Ruegger et al. 2014). qPCR with species-specific primers and probes can provide an accurate and sensitive method for quantification of individual species as well as microbial community populations. Besides, for quantitative purposes, qPCR is more reliable than other methods such as T-RFLP, DGGE/TGGE, and FISH (Ott et al. 2004). Therefore, in the current project (paper IV), we developed a TaqMan MGB qPCR assay targeting the 16S rRNA gene of O. scatoligenes SK9K4T to quantify the O. scatoligenes species in the pig gut. Meanwhile, sequencing of partial 16S rRNA amplicons by Illumina’s MiSeq platform was performed in combination with qPCR to investigate the relative abundance of Olsenella of pig gut microbiota (paper VI).
The use of Taqman-MGB qPCR enabled specific enumeration of the skatole-producing bacterium O. scatoligenes in pig hindgut. We found that O. scatoligenes number were higher in colonic digesta compared to cecal digesta (6.00 vs. 5.12 log cells/g), in males compared to females (5.25 vs. 4.92 log cells/g in cecal digesta; and 6.23 vs. 5.96 log cells/g in colonic digesta); and in young pigs compared to old pigs (5.52 vs. 4.71 log cells/g in cecal digesta; and 6.31 vs. 5.89 log cells/g in colonic digesta). Since the bacterial population of the pig cecum and colon is at least 1010 to 1011 cells/g wet digesta (bacteria is twice as dense in the colon as in the cecum (Butine and Leedle 1989)), we estimated that O. scatoligenes accounted for less than 0.01% (105 – 106 cells/g) of the total population of the hindgut microbiota, which is in agreement with the finding by Jensen and Jensen (1998). Moreover, data from 16S rRNA gene sequencing showed that Olsenella comprised 0.06% of the total microbiota, indicating that Olsenella is not a dominant genus of pig gut microbiota (paper VI). This result is in line with other studies (Kim et al. 2011; Haenen et al. 2013), showing 126
relative abundances of Olsenella is less than 1%.
11.3. Butyrate-producing bacteria
A major metabolic function of gut microbiota is the anaerobic fermentation of dietary fiber in the colon, leading to production of short-chain fatty acids (SCFAs), mainly acetate, propionate and butyrate. Butyrate, in particular, has been identified as the major energy source for colonic epithelial cells and has multiple beneficial effects via its regulatory effects on gene expression (Flint et al. 2008). Butyrate may protect against cancer and ulcerative colitis, and has been associated with preventing the development of insulin resistance and obesity in mice (Berni Canani et al. 2012). Despite the likely importance of butyrate-producing bacteria to the host, the population structure and the taxonomy of these bacteria have not been extensively explored in pigs.
The posible role of butyrate-producing bacteria in skatole levels in the hindgut of pigs and thereby in boar taint was proposed by Claus et al. (2003), who indicated that high levels of butyrate in the hindgut, observed after feeding raw potato starch, reduce skatole production via inhibition of colonocyte apoptosis. In humans, the most important butyrate producers appear to be Eubacterium/Roseburia spp., which belong to the clostridial cluster XIVa (Lachnospiraceae) and Faecalibacterium prausnitzii, which belongs to the clostridial cluster IV (Ruminococcaceae) of the Firmicutes phylum (Duncan et al. 2004; Louis et al. 2010). In pigs, Megasphaera (M.) elsdenii appears to be a predominant butyrate producer (Levine et al. 2013). By testing of 122 OTUs, which represent 2,762 isolates from digesta and feces of finishing pigs, we found that the abundance of the cultivable butyrate-producing community was 8.4%, 11.7%, 17.9%, and 7.0% in the ileum, cecum, colon and feces, respectively (paper V). Butyrate producers within clostridial clusters IX (Megasphera and Acidaminococcus) and XIVa (Coprococcus/ Eubacterium/ Roseburia) were the most abundant.
11.4. Effect of dietary fiber on skatole production
In general, the addition of certain DF (e.g FOS, inulin, pectin and RPS, and various feed ingredients rich in DF e.g. (chicory roots, jerusalem artichokes, sugar beet pulp, and lupines) to the diet of growing pigs has been shown to reduce skatole formation in the large intestine (Wesoly and Weiler 2012; Bilić-Šobot et al. 2014; Jensen et al. 2014). Chicory roots and Jerusalem artichokes, which are rich in inulin, have been shown as the most effective in reducing skatole production, though (Rideout et al. 2004; Hansen et al. 2006; Byrne et al. 2008; Kjos et al. 2010; Øverland et al. 2011;
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Vhile et al. 2012; Zammerini et al. 2012). Nevertheless, the mechanism underlying the skatole reduction effect of these ingredients is unclear. An in vivo study using entire male pigs fitted with a catheter in the caecum was conducted to explore the effect of chicory roots and exogenous butyrate on skatole production and gut microbiota (paper VI). As expected, we found that chicory roots supplementation resulted in lowest numerical levels of skatole in both feces and plasma.
11.5. Effect of dietary fiber on microbial activity
High levels of DF are often seen to increase microbial activity in the GI-tract (Bach Knudsen et al. 1991; Jensen and Jørgensen 1994). However, Heinritz et al. (2016) reported lower total bacterial numbers in the pigs fed a low-fat/high-fiber diet. Reduced fecal microbiota diversity (characterized as reduced richness and evenness) was also observed in our study in pigs exposed to a high chicory roots diet (paper VI). Varel et al. (1982) noted that there was initially a decrease in the bacterial population of the pig microbiota when the pigs were fed with a high-fiber diet. The microbial population, however, increased after continuous fiber-feeding for 12 weeks, suggesting that the microbiota is initially suppressed when exposed to a high-fiber diet, followed by an adaption process and a reestablishment of itself.
Regarding the DF fermentation products, neither butyrate proportion nor total SCFA levels in the feces were affected by the high level of dietary chicory roots supplement. In line with our study, Rideout et al. (2004) and Vhile et al. (2012) did not observe differences in fecal butyrate content in response to dietary supplement of INF. On the contrary, in other studies, stimulation of butyrate production has been observed in response to addition of high levels of carbohydrate with low ileal digestibility such as chicory inulin (Jung et al. 2015) and RPS (Claus et al. 2003; Mentschel and Claus 2003). One possible explanation is that SCFA are largely absorbed in the hindgut, thus, the fecal level of butyrate may not be representative for the actual level in the hindgut.
The direct evidence of enhanced incorporation of TRP into microbial biomass in the presence of higher fermentable fiber in the diet is still lacking. However, type and amount of DF is probably a major regulator of microbial activity and fermentation, and hence, on the synthesis of indolic compounds (Knarreborg et al. 2002). Previous studies have suggested that fermentable carbohydrates stimulate the saccharolytic activity of the microbiota at the cost of proteolytic activity (Jensen and Jensen 1998; Knarreborg et al. 2002). As microbial growth is stimulated by fermentable carbohydrates, the demand for amino acids, including TRP, for microbial protein synthesis increases with the dietary fermentable fibre level, and thus, more TRP is incorporated into microbial biomass,
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leaving less TRP to be converted to skatole.
11.6. Effect of dietary fiber on skatole-producing bacteria
So far, bacteria that have been confirmed as skatole producers belong to Clostridium, i.e., C. scatologenes and C. drakei, isolated from soil and sediment, respectively. We reported Olsenella. scatologenes (isolated from pig gut) and O. uli (isolated from either human gingival crevices or periodontal pockets) to be able to produce skatole indirectly from TRP, via the intermediator IAA (Li et al. 2015). Vhile et al. (2012) reported that the skatole-reducing effect of Jerusalem artichoke coincided with the reduced growth of C. perfringens, which can potentially be used as an indicator of the number of Clostridium genus present. Thus, these authors hypothesized that adding high levels of dietary fermentable fiber results in reduced growth of the skatole-producing bacteria Clostridium in general in the hindgut of pigs (Vhile et al. 2012). Øverland et al. (2008) also hypothesized that organic acids, which have an antimicrobial effect, would suppress skatole-producing bacteria and thereby reduce the skatole formation in the hindgut of entire male pigs.
In the present project, we found that the skatole-reducing effect of chicory roots did not coincide with the reduced number of the skatole producer O. scatologenes. In contrast chicory roots seemed to increase O. scatoligenes proliferation (paper IV and VI). This indicated that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Thus, the abundance of O. scatoligenes in the hindgut is not an appropriate indicator of boar taint. In agreement with our study, an increased proliferation of Olsenella genus by DF has also been revealed by other studies (Haenen et al. 2013; Mao et al. 2015). Further, in contrast to the results of Vhile et al., (2012), no inhibitory effect of chicory roots on Clostridium in feces was observed in our study (paper VI).
11.7. Effect of dietary fiber on butyrate-producing bacteria
In agreement with Levine et al.( 2013), we confirmed that M. elsdenii is a predominant butyrate producer in the pig gut microbiota (paper V). Previous studies showed that feeding a diet with chicory roots or sweet lupins to pigs results in increased colonic abundance of M. elsdenii (Mølbak et al. 2007; Liu et al. 2012a). Further, feeding a Jerusalem artichoke-rich diet reduced the level of C. perfringens in both colon and rectum (Vhile et al. 2012). In line with previous findings, we found that high-fiber diet was correlated with lower abundance of C. perfringens, and C. rectum in the
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small intestine and Acidaminococcus fermentans in the hindgut, but higher abundance of M. elsdenii in the hindgut (paper V). Our in vivo study showed that Megasphaera was numerically higher, whereas Roseburia was significantly lower when chicory root diet was fed to entire male pigs (paper VI). Therefore, chicory roots may selectively promote the growth of specific butyrate-producing bacteria such as M. elsdenii in pig hindgut. However, the skatole reducing effect of chicory roots was not associated with a general increase in the population of butyrogenic bacteria.
11.8. Effect of exogenous butyrate on skatole production
Intra cecal infusion of physiologically relevant amounts of butyrate did not decrease skatole levels in feces or plasma; in contrast, higher values of skatole were detected (paper VI). In line with our study, Øverland et al. (2008) reported that addition of inulin-coated butyrate to the feed, in which the butyrate is expected to be released in the hindgut, has not shown to have an effect on skatole levels in colon, plasma, or adipose tissue. In fact, the effect of cecal butyrate infusion on intestinal cell proliferation and apoptosis is controversial (Kien et al. 2007; Kien et al. 2008). The increased skatole concentrations found by butyrate infusion in the present study may be due to increased cell apoptosis either in the small intestine or the large intestine, leading to more TRP from cell debris being available for skatole formation. The current results indicate that exogenous butyrate alone does not prevent skatole formation; in fact, a tendency towards the opposite was observed.
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12. Conclusions
This PhD project provided new knowledge on the skatole-producing bacteria inhabiting the hindgut of pigs, their metabolism, genomic information, and established quantitation methods to monitor their density in the hindgut of pigs. This study further explored the mode of action behind the reducing effect of DF on skatole production. This may aid finding strategies to reduce the skatole production in the hindgut of pigs and thereby its deposition in fat with the consequent reduction of boar taint. The overall conclusions are summarized below. i) Cultivable skatole-producing bacteria in the pig GI-tract seem to be limited to one species, i.e., O. scatoligenes. Olsenella is not a predominant genus in pig hindgut; and O. scatoligenes makes up < 0.01% of the total bacterial population in the pig hindgut. ii) In pigs, the most important butyrate producers appear to be Megasphera and Acidaminococcus within clostridial cluster IX; and Coprococcus/Eubacterium/ Roseburia within cluster XIVa. M. elsdenii seems to be a predominant butyrate producer in the pig gut. iii) High levels of dietary chicory roots promoted O. scatoligenes proliferation in the hindgut. This indicated that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the hindgut. Thus, the abundance of O. scatoligenes in the hindgut is not an appropriate indicator of boar taint. iv) Intracecal infusion of butyrate alone did not prevent skatole formation in the pig hindgut. v) Chicory roots may selectively promote the growth of specific butyrate-producing bacteria such as M. elsdenii in pig gut, but the skatole reduction effect of chicory roots is not associated with a general increase in the population of butyrate-producing bacteria. vi) The reducing effect of chicory roots on skatole production in the hindgut of pig is most likely due to more TRP incorporated into microbial biomass.
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13. Perspectives
In the current study, we have purified and characterized a novel skatole-producing bacterium, O. scatoligenes, from the pig gut. So far, the first one, and therefore it is the only gastrointestinal skatole-producing bacterium that can be served as a model organism in studies on skatole metabolism. This may aid elucidating processes and mechanisms for reducing skatole production in pig gut and eventually, deposition of skatole in back fat of entire male pigs. Further, we have established a Taqman-MGB real-time PCR assay to quantify O. scatoligenes in the pig hindgut and proved that the known effect of chicory roots on skatole in the hindgut is not by inhibiting the growth of this skatole-producing bacterium.
Instead, suppression of the enzymatic activity of skatole synthesis may be responsible for the reduced skatole production by DF. In order to verify this assumption, candidate genes involved in the skatole synthesis pathway need to be identified. This goal can be achieved by conducting comparative genomic analysis with closely related Olsenella species, including both, those able to produce skatole (e.g. O. scatoligenes and O.uli) and those unable to do so (e.g. O. profusa and O. umbonata). However, in this project, we only obtained the draft genome of O. scatoligenes. It is possible that the relevant regions regarding skatole metabolism are absent or not correctly represented in a draft genome. Therefore, the first step to identify genes involved in skatole synthesis is to close the gaps of the O. scatoligenes draft genome before conducting comparative genomic analysis. The advantages of characterizing the genes involved in the skatole synthesis pathway are not limited to identifying more skatole-producing bacteria by screening of publicly available (meta)genomic data using the marker gene(s). It will also aid to find substances to inhibit skatole synthesis activity, or to completely block the synthesis pathway by genome-editing techniques.
Moreover, we have ruled out two hypotheses for the mode of action of the skatole-reducing effect of chicory root in the current study. We have found that this effect is neither via inhibition of cell apoptosis by butyrate, nor via suppression of skatole-producing bacteria in the pig hindgut. However, direct evidence lacks regarding the third hypothesis on the impact on the microbial activity, that is, more amino acids (including TRP) are incorporated into bacterial biomass due to the presence of high fermentable fiber and a corresponding suppressed proteolytic conversion of TRP into skatole. The 16S rRNA marker gene metagenomic study allowed elucidating the composition and diversity of the gut microbiota as affected by diet, but this technique does not offer
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information on metabolic changes of the gut microbiota. Application of meta-omics technologies, metatranscriptomics, in particular, may provide insight into microbial community activity, and thus aid to elucidate the third hypothesis.
The precursor TRP is first deaminated to IAA, which is then further decarboxylated to skatole. In order to elucidate whether exogenous (from dietary) or endogenous (from intestinal cell debris) TRP is the main sources for microbial production of skatole, in vivo feeding studies or in vitro fermentation studies with stable isotope labeled tryptophan can be performed to monitor skatole synthesis in the pig hindgut. Besides, labeled TRP will allow tracing the proportion of TRP incorporated into microbial biomass when sufficient carbohydrate is available in the pig hindgut.
Together, these efforts will contribute to elucidating the skatole metabolic pathways, hence facilitating the reduction of skatole production in the gut and, thus, boar taint in pigs. Additionally, a better understanding of the mode of action of the skatole-reducing effect of DF will be improved.
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