Functional Analysis of Changes in Gut Microbiota Genetic Potential in Broilers Supplemented with 2% Oregano

Throughout the world, poultry are a major and growing source of high-quality protein, as they outperform all other terrestrial meat production systems in water, feed, carbon, and land use efficiency. A challenge for intensive production systems has been the potential to introduce high pathogen loads and stresses on the animals. Such challenges have traditionally been managed with the assistance of antibacterial growth promoters. However, the poultry industry in Australia, and in many other regions, is working to reduce the use of in-feed antibiotics and alternative products are required to maintain health and productivity. We can look to nature for alternative antimicrobial compounds. Bacteria can infect almost all organisms, and, in response, many hosts have evolved the ability to produce antibacterial compounds to help them fight off pathogenic bacteria. Plants, especially medicinal herbs and spices, produce a range of antibacterial phytochemicals. Phytobionts such as oregano, clove and cinnamon’s antimicrobial ingredients, are showing comparable, if not better, results than subclinical antibiotics in pathogen control. There are multiple reports on their effect on individual poultry pathogens such as Salmonella, and on performance. Lately, more reports are becoming available on their effect on whole bacterial communities. However, the changes they introduce to the functional capabilities of microbiota, i.e., what microbiota can do for the host, are equally important and give a very different picture to that produced by simple taxonomic profiling of microbiota. Here we present the first insights into the effects of oregano on the functional capabilities of intestinal microbiota in broilers.

Dried oregano (Turkish, Saucy Spice Company, NSW, containing 2-3% carvacrol) was used to make a powder with an average particle diameter of 10μm. Chicken starter diet (Red Hen, Laucke Mills, Australia) with no antimicrobials or coccidiostats was used for the duration of the trial. The oregano was mixed into the feed at a 2% inclusion rate (0.02 kg/kg w/w). The study was approved by the Animal Ethics Committee of Central Queensland University under approval number 0000020312. One day old Ross Broiler 308 birds (Bond Enterprises, Toowoomba) were randomly distributed into two groups with n=12 per treatment. All birds were fed ad libitum and had unrestricted access to drinking water. Birds were individually tagged using leg bands and weighed every week for 42 days. Fresh faecal material was collected for each bird. The contents from the jejunum, ileum and caecum intestinal contents, together with faecal samples were taken for sequencing analysis.

DNA was extracted from samples using a previously described protocol (Bauer et al., 2019). The 16S rRNA gene sequencing library preparation and amplification followed the manufacturer's protocol (Illumina Inc., San Diego, CA, USA). Sequencing was conducted on the Illumina MiSeq platform using 2x300 bp paired-end sequencing according to the manufacturer's protocol (Illumina Inc., San Diego, CA, USA). The microbial communities of each sample were initially analysed using QIIME. Phred quality threshold had a minimum of 20. OTUs were picked at 97% similarity using UCLUST (Edgar, 2010). The PICRUSt algorithm (Langille et al., 2013) was used to predict and enumerate genetic functional categories. The sequence data are publicly available at the MG-RAST database under library accession number mgl745316 and project ID mgp89580.

We predicted the functional abilities of microbiota using the KEGG database to compare microbiota of control and 2% oregano treated birds. Adonis multivariate analysis using Jaccard distance showed significant functional differences (Adonis P< 0.05) (Figure 1). The data show that oregano supplementation reduced the abundance of genes in functional categories involved in a number of diseases such as bladder cancer (P=5e-4), prostate cancer (P=0.045), prion diseases (P=0.007) and Shigellosis (P=2.3e-4). The microbiota from oregano supplemented birds had a lower abundance of genes involved in bile secretion (P=5e-5), bacterial motility proteins (P=0.018), flagellar assembly proteins (P=0.023), bacterial secretion (P=0.012) and bacterial invasion of epithelial cells (P=2e-4). Although there were no differences in weight throughout the lifetime of the birds, a range of metabolic functional related genes, such as carbohydrate digestion and absorption (P=1.9e-3), vitamin B6 metabolism (P=0.028), fatty acid metabolism (P=0.001), and steroid hormone biosynthesis (P=0.025) were reduced in oregano supplemented microbiota. Therefore, the data suggest that oregano changes the microbiota structure to reduce bacterial species with the genetic potential for motility, flagella and secretion and their ability to invade epithelial cells, as well as reducing functional capability to contribute to a range of diseases. However, these health beneficial functions come with the cost of reducing microbiota metabolic and digestive ability.

Oregano supplementation in the current study significantly reduced the presence of genes involved in flagellar assembly, bacterial motility and bacterial invasion of epithelial cells. It was previously reported (van Alphen et al., 2012) that oregano’s main antimicrobial, carvacrol, inhibited the motility of Campylobacter jejuni without affecting bacterial growth. This is of significance because it indicates that oregano can reduce pathogenic potential in a way that is not detectable using current culturing or microbiota sequencing methods since the bacterial numbers do not have to be affected. Sub-inhibitory concentrations of carvacrol were shown to block motility and invasion of epithelia by C. jejuni via interfering with flagella function (van Alphen et al., 2012); the reduction of flagellar genes in the oregano supplemented microbiota was also significant in the current study. Additionally, carvacrol has been shown to induce heat shock protein 60 and inhibit the synthesis of flagellin in Escherichia coli (Burt et al., 2007). Oregano essential oil also shows anti-Giardia activity via disruption of its adherence (Machado et al., 2010). Moreover, oregano essential oil abolishes Salmonella enterica serovar Enteritidis in pre-formed biofilms on stainless steel via multi-target action mode on bacterial cell membrane (Lira et al., 2019) and is efficient against antibiotic-resistant Salmonella enterica (Moore-Neibel et al., 2013). The anti-Salmonella activity is in agreement with our functional analysis findings that oregano reduces motility, bacterial secretion, and invasion of epithelia, all major Salmonella weaponries.

The anticancer activity of oregano reported in our functional analysis is aligned with previously reported anticancer properties of carvacrol in breast, liver, and lung carcinomas (reviewed in Sharifi-Rad et al., 2018), coupled with strong antioxidant and anti-inflammatory capabilities (Sharifi-Rad et al., 2018).

Figure 1 - Functional analysis of changed microbiota genetic potential due to influence of oregano performed in PICRUSt.

Our results indicate that oregano reduced the abundance of genes involved in digestion of carbohydrates and fats as well as steroid hormones and bile. The reports on oregano helping digestion (Reyer et al., 2017; Sharifi-Rad et al., 2018) are opposite to our findings; however, even if significantly altered, the reduction of the digestion-related genes in intestinal bacteria will not necessarily result in measurable reduction in the host digestion, but could also reduce growth of bacteria. Host digestive efficiency depends on a range of host factors, feed composition, and bacterial contribution, to name a few (Swallow, 2003). On the other hand, oregano also reduced the abundance of bacterial genes involved in host bile secretion which strengthens the possibility that oregano can indeed slow down the host digestion process. Our study focused on genomic analysis and did not have the power to make conclusions regarding performance; however, the data on ileum histology on these same birds (not shown) demonstrated no differences in villus height or crypt depth.

Oregano in feed significantly reduced the abundance of bacterial genes involved in steroid hormone biosynthesis. Intestinal microbiota can produce biologically active molecules including sex hormones, by acquiring genes from the host via bacterial transformation. Although it is unlikely that the host depends on microbiota for steroid production, faecal transplants from male to female mice resulted in female mice producing testosterone at equivalent concentrations to that of males (Markle et al., 2013). This study was the first to show microbiota producing human host hormones. There are also other indications that oregano can induce oestrogenic responses in vitro and can also show oestrogen-like activity (Wielogorska et al., 2019), thus influencing steroid hormone balance. Oregano could be a suitable natural product to treat conditions related to lower oestrogen production (Beck and Hansen, 2004). The reduction in steroid synthesis capability also opens a question of possible oregano interactions with stress response via regulation of another steroid hormone – cortisone. The effects of oregano on the genetic capability of microbiota may be of higher or equal importance as the direct effect on the specific bacterial taxa abundance.