Abstract

The microbiome is a multifarious and dynamic ecological element, in which different species such as bacteria, fungi, archaea, protozoa, and viruses are in continual flux. They can alter host development, the physiology, and systemic defenses that play a great role in food producing animals. Perturbation to the microbiota or dysbiosis affects greatly the host’s normal physiology and may cause different diseases in the host. Microbiomes can be studied in different ways including different molecular techniques and omics technologies. Microbiome engineering is an experimental method that improves host performance by artificially selecting for microbial communities with specific effects on host fitness. Different methods are used in microbiome engineering and these include synthetic biology, modulating the microbiota, antibiotics, feed enzymes, prebiotics, probiotics, fecal microbiota transplantation, horizontal gene transfer, acceptance, and maintenance of foreign DNA, and genome editing. All these methods participate in the manipulation of the genomic content of microbiomes. This technique has different advantages including treatment mental health problems, modulating host immunity, improving nutrition, modifying physiology of the hosts and the characteristics of the ecosystems where they reside, modulates the pathogenesis, progression, and treatment of diseases, improving agricultural productivity, controls the variability for the efficiency of feed utilization in ruminants. Therefore, engineering microbiomes has a great advantage in improving the production systems of livestock.

1. Introduction

Livestock production has significant economic importance and reinforces food security in different parts of the world. Ruminants play a great role in the production of meat and milk which are major sources of protein for humans ¹. The digestion of feed such as straw, hay, silage, and grass, and the production of enteric methane, carbon dioxide, and hydrogen, which are the main electron acceptors and donors of the ecosystem is vital purposes that could be operated by having a thorough understanding of the rumen microbiome ^{2, 3}. This manuscript aims to review different methods of engineering livestock microbiomes important for livestock production improvements.

2. Microbiome

The microbiome is a complex and dynamic ecological unit, in which different species are in continual flux. Many tools from theoretical ecology are used in considering the impression and forecasting the dynamics of the microbiota ^{4, 5, 6}. This ecological unit can be considered as a halobiont organism that populates different niches in mammals and interacts with the host, in most cases, in a symbiotic manner such as during metabolic functions by its different individual microbial members ² and the digestion of feed, or modulating the immune response ^{7, 8}.

Microbiomes, host-associated microbial communities, in animals include bacteria, fungi, archaea, protozoa, and viruses ^{9, 10}. They can alter host development, physiology, and systemic defenses that play a great role in food producing animals ¹⁰. They contain a hundredfold more genes than host genomes ¹¹. Perturbation or disturbance to the microbiota can result in an extensive modification to the hosts and environment, such as reduced host fitness and health problems ¹².

3. Rumen Microbiome

The rumen microbiomes are numerous, complex, and diversified communities of interacting microorganisms inhabiting the rumen, including bacteria, protozoa, fungi, and archaea ^{3, 13}. Firmicutes and Bacteroidetes are the main bacterial phyla found in the cattle’s rumen. Firmicutes are mostly greater in relative abundance in predominantly forage-based diets, while Bacteroidetes are more abundant in concentrate feeds ¹⁴. They are classically succeeded in abundance by Actinobacteria, Tenericutes, and Proteobacteria ¹⁵. At the genus level, Prevotella is most common and there is a wide range of functional capacities of species within the genus. Since there is no known normal rumen microbiome, it is typically examined under specific conditions, such as diet, production stage, or illness. The one that is normal on one nutrition or in one condition may not be normal in another. Therefore, these factors affect microbiome composition in ruminants ¹⁶. Genus Lactobacillus consists of a different class of microbes; among them are mostly the members of human gut microflora. Probiotic L. acidophilus, L. brevis, L. casei, L. gasseri, L. johnsonii, L. plantarum, L. reuteri, and L. salivarius are the most important and common gut bacteria ¹⁷.

4. Dysbiosis

A microbiota is a community of microbes that naturally associate closely with a multicellular host ¹⁸. These communities of bacteria and fungi interact extensively with the host in often symbiotic or commensal relationships that can cause a disease-associated state known as dysbiosis when perturbed or unbalanced ¹⁹. Compositional perturbations of the microbiota (dysbiosis) have been associated with diseases including obesity ²⁰, diabetes ²¹, colorectal cancer ²², allergies ²³, and underperformance ^{8, 24}.

Maintaining compositional and functional stability within the gut microbiome is essential to the host’s health as demonstrated by dysbiosis detected at the onset of non-pathogenic chronic diseases ^{25, 26}, like Crohn's disease, where the gut microbiome has a significant reduction in important Bifidobacteriaceae populations while exhibiting an increase in groups containing potential pathogens including Enterobacteriaceae, Fusobacteriaceae, Neisseriaceae and Pasteurellaceae ²⁷. Transcriptome data can be used to explore the results of perturbations and environmental factors on the function of the gut microbiome. Additionally, it can consider microbiome functional alteration before a compositional change occurs ^{28, 29}. The reduction of the bacterial population in the gut has been associated with obesity ³⁰, and the increased presence of species in the reproductive system has been associated with bacterial diseases of the reproductive system ³¹.

5. Studying the Microbiomes

Microbiomes can be studied in different ways. Some of them are DNA sequencing technology and Southern blotting techniques ^{2, 32}, PCR-based massive parallel sequencing technologies ³³, Shotgun metagenome sequencing, and technological advances in RNA-sequencing are also used to study unculturable microbes ³⁴, and the integration of transcriptome analysis enhanced metabolic pathway predictions ¹⁷. Quorum-sensing molecules enable the regulation of gene expression in response to fluctuations in cell population density ³⁵. Metagenomic, genomic, and 16S rRNA sequencing of different samples have given rise to a plethora of genomic information and the means of studying the composition of the gut microbiome ^{2, 18}.

Different omics methods expand our knowledge in understanding the molecular mechanism of interaction between probiotics and the gut environment ¹⁷. Determining metagenome composition might assist as a phenotype proxy for feed efficiency in livestock species, but a large reference population needs to be established with metagenome information that serves the purpose of genomic selection on feed efficiency. It must imply lower costs than phenotyping for direct dry matter intake ⁸. Metatranscriptomics and DNA-based shotgun metagenomic sequencing ^{34, 36, 37}.

6. Microbiome Engineering

Microbiome engineering is an experimental method that improves host performance by artificially selecting for microbial communities with specific effects on host fitness. It applies multigenerational, artificial selection upon hosts that vary in microbiome content affecting the host trait ¹⁰, and it also deals with leveraging fundamental scientific principles and quantitative design to create microbiomes that perform desired functions ³⁸.

In engineering microbiomes, microbial compositions can be altered to improve host phenotypes and benefit ecosystems. The animal microbiome is composed of diverse microbial communities and the composition of the microbiota correlates with the hosts’ growth and health ^{12, 23}. Many studies have largely focused on understanding the gut microbiota in representative animals including mice, rats, broiler, swine, and cow. Although mice and rats are the most commonly studied animals, they have been mainly used as animal models for the engineering of the human microbiome. In this section, we emphasize microbiome engineering of animal livestock for improving animal health and agricultural productivity ^{39, 40}. Manipulating the microbiota directly affects the health of the host ⁴¹.

Identifying conditions for the proper isolation and culturing of individual species are the prior steps in genetic tool development ⁴¹. Matrix-assisted laser desorption time of flight or 16S rRNA amplification and sequencing are used to identify unassigned species within metagenomic sequence databases. High-throughput isolation and screening will greatly increase the number of culturable species from the gut microbiota ⁴². The next steps to creating genetically tractable strains will be to (a) transport and maintain exogenous DNA, (b) enable predictable expression of heterologous genes, and (c) regulate endogenous genetic materials ⁴³. Emerging technologies including engineered organoids derived from stem cells, high-throughput culturing, and microfluidics assays allowing for the introduction of novel approaches to improve the efficiency and quality of microbiome research ³³.

Notable developments in the technique include synthetic cell-to-cell communication ⁴⁴, complex and large-scale genetic circuits ⁴⁵, and clustered regularly interspaced short palindromic repeats (CRISPR)-based regulation ⁴⁶. For the field of microbiota engineering, synthetic biology provides a means to study structure-function relationships among microbiota and engineer novel biotic therapeutics. The incorporation of artificial genetic pieces of machinery increases the capabilities of the engineered microbe to sense, record, and respond to its environmental conditions ^{47, 48}. The success of the designed function depends on the availability of genetic tools. This technique is limited to model laboratory strains and a select few host-associated species. However, recent advances in expanding the genetic tools available for other non-model-host-associated microbes have opened the door for new and emerging applications for engineering synthetic biotics ⁴¹.

Engineering the microbiome might enable studying the contribution of individual microbes and generating potential therapies against metabolic diseases such as phenylketonuria and chronic kidney disease, inflammatory, and immunological diseases, among others ^{25, 28}. Methods for probing the microbiome include fecal microbiota transplantation and the use of antibiotics, probiotics, and prebiotics; these can produce broad alterations in the microbiome and its reciprocal interactions with the immune system. Novel tools are still required to exactly reprogram microbial communities to improve health and disease outcomes ³⁵. Microbiota manipulation using CRISPR-Cas engineered bacteriophage can selectively remove target strains from a community based on the presence of target DNA sequences ⁴⁹.

Synthetic biology is a rapidly growing field that aims to design and achieve programmed cellular behavior by using natural and synthetic biological components. This type of forwarding engineering has created numerous biotechnological advances from chemical product biosynthesis to complex therapeutics ^{50, 51}. Engineering indigenous microbiota community members and non-resident probiotic bacteria as biotic diagnostics and therapeutics that can probe and improve the health of an organism ⁴³.

Synthetic biologists use the term chassis to refer to the type of cell that harbors and maintains the DNA constructs needed for a particular function. Decisions about which chassis to use for interactions with the microbiome rest on viability, colonization, localization, and genetic tractability ⁵² or the use of green fluorescent protein to track E. coli location and persistence in the rat intestine ^{53, 54}. Localization refers to the specific region of the gut affected by the disease. For example, Bacteroides spp., which localize in the cecum and colon, might be used to treat ulcerative colitis, which only affects the large intestine; and Lactobacillus spp., found in the small intestine, might be used to treat Crohn’s disease, which can affect any part of the gastrointestinal tract ^{52, 55}. Genetic tractability refers to whether the organism can feasibly be genetically modified by transformation, gene expression, activation, or other means ³⁵.

The microbiota is an effective modulator of host immune responses that can be targeted for therapeutic purposes. Antibiotics, dietary interventions, or fecal microbiota transplantation have been explored as tools to reshape the gut bacterial communities and to treat allergic and immune conditions ⁵⁶. Factors affecting microbiome establishment in ruminants include diet, feed additives, and weaning ¹⁶.

Modulating the microbiota includes, (i) Molecule-mediated alteration of dysbiotic microbiota, (ii) in fecal matter transplants (FMTs), the fecal microbiota from a healthy donor is collected and transferred to a recipient, (iii) DNA delivery and transfer through microbes or phages can provide a means of rational in situ microbiota engineering. Future applications could apply in situ transfer of DNA to members of the microbiota to reprogram microbiota functions ^{41, 57}.

Microbial consortia are mutually dependent groups of bacteria (i.e., bacteria that live in symbiosis). Perhaps the most well-known use of consortia for therapeutic purposes is the use of fecal transplantation to treat Clostridium difficile infections ⁵⁸. However, organisms interacting in a bacterial consortium cannot be genetically engineered in situ by conventional methods ⁵⁹. One approach for this may be to create synthetic consortia composed of a known cluster of bacteria. A combination of microbiome engineering tools (such as phages, antibiotics, probiotics, and diet) could be applied, depending on the target species ³⁵.

Antibiotics are commonly used in livestock farming to prevent different bacterial infections ⁶⁰ and promote animal growth but their administration has caused the emergence of antibiotic-resistant bacteria and raised concerns regarding residual antibiotics in meat products. Thus, microbiome engineering of livestock by using feed enzymes, prebiotics, and probiotics to alter the microbiota composition is a suitable alternative to antibiotics utilization for improving animal health in the animal farming industry ⁴⁰. Antibiotic administration can result in drastic, unpredictable changes in microbial composition and elimination of pathogens; however, the mechanism through which the healthy flora repopulates the gut is poorly understood ⁵⁷.

The specific elimination of members of the microbiome by targeted antimicrobials remains a grand challenge in the field. Promising strategies to knock out specific bacteria include bacteriocins ⁶¹ and bacteriophages ⁶². Despite their potential, only two bacteriocins are commercially available (nisin from Lactococcus lactis and carnocyclin A from Carnobacterium maltaromaticum). Engineered bacteriophages are used as antimicrobial agents to treat bacterial infections ³⁵.

Feed enzymes, such as phytase, amylase, non-starch polysaccharide (NSP)-degrading enzymes (e.g., xylanase, β-glucanase, and β-mannanase), proteases and lysozyme, have been used to promote gut health in swine and poultry by improving substrate digestion, increasing production of prebiotics from dietary NSPs, and reducing anti-nutritive factors ⁶³. As a result, the microbiota composition and maturation of gut microbiota have been accelerated. These feed enzymes have positive effects in promoting animal growth and controlling infectious diseases including post-weaning colibacillosis in piglets, swine dysentery, salmonellosis, and necrotic enteritis ⁴⁰.

They are foods, or compounds found in food, that induce the growth of microbial that are helpful ⁶⁴. Another definition of prebiotic is a substrate that is selectively utilized by host microorganisms and confers a health benefit. This definition also applies to prebiotics for animal use ^{35, 65}. Oligo- and mono-saccharides, such as fructo-oligosaccharides, mannan-oligosaccharides, xylo-oligosaccharides, and inulin, are the dominant prebiotics that have been used to alter animal microbiomes. Prebiotics supply enables modification of the animal gut microbiota and results in the production of (i) short chain fatty acids (SCFAs) that reduce luminal pH, provide energy sources, regulate metabolism, and modulate immune systems ³²; (ii) antimicrobial factors that inhibit pathogens; and (iii) prebiotic derivatives that reduce pathogen’s adhesion to host cells ^{24, 29}. Dietary supply of inulin to swine leads to an increase in probiotics, a decrease in less desirable microorganisms, inhibition of pathogens, and accelerated maturation of the bacterial community in the swine gut ^{12, 18}. Likewise, beneficial effects of prebiotics supply have also been detected in broiler chicken ^{3, 6}.

Probiotics are living microbes such as bacteria and fungi, found in nutritive supplements or food ⁶⁴. Their administration is effective for engineering animal microbiomes. The dominant probiotic strains used are Lactobacillus, Bifidobacteria, Enterococcus, Bacillus, and Saccharomyces boulardii. Their administration can be used to treat infectious diseases as well as to promote growth performance. For example, administration of B. subtilis CH16 increased daily weight gain and reduced food conversion rate in broilers ^{14, 22}. In some cases, probiotics are combined with prebiotics to increase probiotic bacteria levels and survival in gastrointestinal tracts ^{8, 15}.

Probiotics generally have a short-term beneficial effect on the gut microbiota ^{32, 36}, promoting the growth of other beneficial microbes, and facilitating host immune response. Members of the lactic acid bacteria group including several species of lactobacilli are commonly regarded as probiotics ³³. Most of them can be isolated from fermented foods and are temporary members of the microbiota that transiently interact with the host and resident microbes ⁶⁵. The consumption of probiotics maintains the balance of the gut flora and also inhibits the growth of pathogenic bacteria. Probiotics can be taken as a promising alternative to antibiotics since they can displace harmful bacteria through various mechanisms. Probiotics synthesize nutrients in the intestine and make essential compounds available for microflora ¹⁷.

Probiotics and prebiotics alter the composition of the microbiota to achieve better results in combination with personalized diet regimens during regulation of the diet-microbiota axis ⁶⁴. Ingestion of them is used as microbial therapeutics to assist or maintain a healthy gut composition ⁶⁵. They are being evaluated for their effectiveness to treat and prevent infectious diseases in other parts of the body where microbial communities present ⁶⁶. Their supplementation is presumed to stimulate the proliferation of beneficial microbes in the gut, but the health benefits of the microbial population distribution changes through such supplementation has not been demonstrated rigorously ⁶⁷.

Procedure in which feces containing microbiota from a healthy donor are transferred into a recipient ⁴¹. In other words, it the process of transferring fecal matter from one or many individuals to another to affect the microbiome of a recipient ⁶⁴, and also the infusion of gut microbiota from a healthy donor into the intestine of a recipient ^{58, 68}. Different infusion methods exist ⁶⁹, and also, a frozen capsulized FMT approach has been proposed ⁷⁰.

It has gained much attention for microbiome engineering to re-establish healthy microbial composition in patients with gastrointestinal diseases. This method involves acquiring faecal material from healthy donors and administering microbiota-laden suspension obtained from the faecal material to the gastrointestinal tract of the patients ^{40, 71}. It also has received widespread acceptance due to its extremely high efficacy in the treatment of refractory Clostridium difficile infection (CDI) ^{58, 68}. The application of FMT in conditions other than CDI has thus gained interest, and particularly so in the treatment of IBD ⁷². Probiotic supplementation and FMT treatments can be effective at augmenting the population, but the mechanisms through which they alter the native microbiota are poorly understood ⁴¹.

It is well known that genetic material is constantly being transferred between members of the microbiota; the transferred material has been termed the mobilome ^{59, 73}. Plasmid transfer has been demonstrated in mouse gut-associated L. reuteri to E. faecium, and E. faecalis to L. fennentum, providing a foundation for in situ engineering technologies. DNA can be transferred to microbes by conjugation or electric or chemical disruption of the cell membrane, inducing natural competence, or by phage transduction ⁴¹.

Acceptance of foreign DNA depends on transport across the cell wall and membrane(s), avoiding rejection by the host’s defense systems, and active replication within the host. Transport into the cell has been achieved by disrupting the cellular wall and membrane (chemical disturbance, electroporation), injecting the DNA (transduction, conjugation), or using already-present machinery within the host cell (natural competence) ^{41, 52}. Often, conditions for transformation need to be optimized even for different strains within the same species. Once inside the cell, the DNA is surveyed by the host cell’s defense systems, which have evolved to eliminate invading DNA. Examples of these systems are restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Restriction-modification systems are estimated to be present in almost all bacterial species and appear to pose the largest barrier to DNA transformation ^{43, 47}.

Genome editing is traditionally accomplished through homologous recombination by the use of selectable markers ⁴¹. It is quite an extraordinary technique because of its capability to alter DNA by utilizing engineered nucleases called molecular scissors. It finds application in diverse areas since the editing is done according to process fitment. The most efficient and simple technique of gene editing has been described as CRISPR-Cas ⁷⁴.

The major gene editing tools like Clustered regularly interspaced short palindromic repeats (CRISPR-Cas) ^{75, 76, 77}, transcription-activator like effector nucleases (TALEN) and Zinc finger nucleases (ZFN) have made it possible. These gene editing tools introduce double strand break (DSB) in the target gene, which is repaired by the error-prone non-homologous end joining (NHEJ) pathway or homology-directed repair (HDR). Zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALEN) are artificial restriction enzymes that edit or cleave the specific target DNA by using zinc finger DNA-binding domain or utilizing TAL effector DNA binding domains, respectively. The progress in gene editing tools and the development of various methods for easy synthesis and assembly of TALENs allows efficient editing at multiple sites ^{17, 41, 78}. Also, Tn mutagenesis has been used as genome editing technologies ³⁵.

The mechanism of editing often involves targeting using a self-designed guide sequence which is complementary to a unique sequence at a site of interest, prompting a break at a site, which is repaired at high rates using homologous recombination, thereby deletion or insertion of desired fragment ^{79, 80}. To design the customized probiotic the single-stranded DNA recombineering (SSDR) can be integrated with CRISPR-Cas to allow fine base changes in the chromosome ⁷⁶. The approach can be followed using a single step where recombineering oligonucleotide and the CRISPR-plasmid can be co-transformed in a probiotic bacterium expressing RecT and Cas9. The CRISPR-plasmid encoding CRISPR-array along with Cas9 and the tracrRNA cleave desired sequence until SSDR make the change ^{80, 81}. Thus, such techniques provide the opportunity to enhance the scope of probiotics to maintain the health issues by making changes at a level of a single nucleotide ¹⁷.

Microbiomes have different importance and contribution in both animals and humans. Some of these include treatment mental health problems ⁸², modulating host immunity ⁸³, modification of host metabolism ⁸⁴, improving nutrition ⁸⁵, resistance to colonization ⁸⁶, and modifying physiology of the hosts and the characteristics of the ecosystems where they reside ⁴⁰. The microbiota also modulates the pathogenesis, progression, and treatment of diseases ^{64, 87}.

Microbiome engineering has established success in an application for the treatment of different diseases and improving agricultural productivity ⁴⁰. Additionally, gastrointestinal microbiomes control the variability for the efficiency of feed utilization in ruminants. Hence, modifying the microbiota composition helps a more sustainable and efficient livestock production to get their products for human feed ⁸. Therefore, engineering microbiomes has a great advantage in improving the production systems of livestock and these techniques should further be developed for the future.

In the ongoing pandemic of the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), that has been responsible for CoV Disease-19 (COVID-19), it was noted that the gut microbiota could influence the course and the disease outcome ⁸⁸. Also, it was demonstrated that the dysbiosis of gut microbiome predisposes people to infection, inflammation, and associated gastrointestinal complications ^{89, 90}.

Evidence of the replacement of commensal/residential gut microbiome with potentially invasive ones could lead to increased infections and inflammation ^{91, 92, 93, 94, 95}.

Therefore, there is an increased belief that application of probiotics in the management of COVID-19 and other infections may favour disease prognosis. Also, the probiotics could prevent the excessive immune response (cytokine storm), reduce inflammation, and prevent microbial multiplication and invasion ^{96, 97, 98}.

7. Conclusion

Engineering microbiomes in livestock has many advantages. There are different techniques used in the engineering of microbiomes. When microbiomes are manipulated, they are used for treating of mental health problems, modulating host immunity, improving nutrition, modifying physiology of the hosts and the characteristics of the ecosystems where they reside, modulates the pathogenesis, progression, and treatment of diseases, improving agricultural productivity, controls the variability for the efficiency of feed utilization in ruminants. But when perturbated it affects the normal physiology of the animals and may cause different diseases in the host. Therefore, the growth of engineering microbiomes is the crucial method for the improvement of livestock production.

Abbreviations

CDI: Clostridium difficile infection; CRISPR: Clustered regularly interspaced short palindromic repeats; DNA: Deoxyribonucleic Acid; DSB: double strand break; FMT: Faecal Microbiota Transplantation; HDR: Homology-directed repair; IBD: Inflammatory bowel disease; NHEJ: error-prone non-homologous end joining; NSP: Non-starch Polysaccharide; PCR: Polymerase chain reaction; RNA: Ribonucleic acid; SCFAs: Short chain fatty acids; SSDR: single-stranded DNA recombineering; TALEN: transcription-activator like effector nucleases; ZFN: Zinc finger nucleases

Ethical Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Availability of Supporting Data

Not applicable.

Competing Interests

The author declares that there are no competing interests.

Funding

This manuscript has no fund.

Acknowledgements

The author (Gamechu Berhanu) would like to acknowledge staffs of Dambi Dollo University for their assistance in gathering valuable information for writing this review.

References