Developing a “Super Microbe” to Reduce Antibiotic Use in Beef Cattle

Project Summary

Developing a “Super Microbe” to Reduce Antibiotic Use in Beef Cattle

Principle Investigator(s):: Alison C. Neujahr, Samodha Fernando
Institution(s):: Department of Animal Science, University of Nebraska-Lincoln
Completion Date:: November 2020

KEY TAKEAWAYS

Genes allowing for inhibition against liver abscess causing pathogens from non-Generally Regarded as Safe (GRAS) were successfully identified and inserted into a GRAS-approved bacteria which can be utilized in a beef production setting.
CRISPR-Cas9 genome editing tools together with transposon mutagenesis can be used to develop novel direct fed microbials (DFMs) with increased capacity for pathogen inhibition by using naturally occurring genes, ultimately improving cattle health and productivity.

BACKGROUND

Antibiotic use in the beef industry has come under great scrutiny. As a result, the need to reduce prophylactic use of antibiotics and develop new technologies to improve animal health is greatly needed. One of the largest animal health related issues in the current beef industry is liver abscesses. Liver abscesses cause an economic loss to the producer and the packer as liver abscesses are the leading cause of condemnation in the United States. While the liver is not a significant loss, abscesses formed results in reduced animal performance and carcass yield via reduced feed intake, reduced weight gain, decreased feed efficiency, and decreased carcass dressing percentage. Currently, the most effective method for controlling liver abscesses is the use of antibiotics belonging to the macrolide family named tylosin. Even with the use of antibiotics, 10-20% of fed cattle are affected and form liver abscesses. The current hypothesis is that cattle fed highly fermentable carbohydrates leads to dysbiosis in the rumen microbiome leading to lactic acidosis, which in turn leads to damage of the rumen epithelium resulting in opportunistic pathogen Fusobacterium necrophorum necrophorum (F. necrophorum) to enter the liver through the portal vein leading to liver abscesses. However, apart from prophylactic use of antibiotics, a proven strategy for reducing liver abscesses has not been developed.

Direct fed microbials (DFMs) are potential promising alternatives to reduce antibiotic use in beef cattle production and to improve animal health, well-being, and productivity. However, many of the strains with the highest potential to reduce antibiotic use are found in non-GRAS (generally accepted as safe) organisms. Additionally, since a single microbe cannot inhibit all pathogens, DFM cocktails need to be used to be effective on a broad spectrum. Many isolates capable of inhibiting F. necrophorum have been previously identified, some of the most effective isolates being from non-GRAS approved strains. As such, we hypothesized that introducing naturally occurring inhibitory genes from non-GRAS approved organisms to a GRAS approved microbe, Bacillus pumilis, will help develop a single microbe capable of decreasing multiple pathogens with multiple benefits towards increasing animal productivity.

Methodology

DNA was extracted for Whole Genome Sequencing of naturally occurring non-GRAS isolates and prepped for sequencing on the Illumina MiSeq platform. Raw reads were used to identify potential bacteriocins produced to gain insight into which gene or genes within this strain can cause inhibition of F. necrophorum and Streptococcus bovis (S. bovis). CRISPR/Cas9 assays were used for gene knockouts (KO) from the identified potential genes from the wild type (WT) Aneurinibacillus migulance strain FA2B. FA2B-knockout (KO) isolates were individually picked and used for functional screening and PCR analysis to confirm the loss of inhibition towards F. necrophorum and S. bovis. Bacillus pumilus was used to insert identified genes to create a mutated bacteria which was investigated for insertion of gene using PCR and inhibition against S. bovis.

Findings

A draft genome was constructed with greater than 10X coverage of the A. migulance strain FA2B. The resulting genome was screened against Bagel4, bactibase, and antiSMASH to identify potential bacteriocins. This analysis identified 5 genes of interest that included 3 bacteriocin genes, one terpene and one T3PKS. These five genes were identified as potential candidates for inhibiting S. bovis. As such, CRISPR-Cas9 was utilized to KO all five genes, individually, to facilitate the identification of which gene or gene combination was needed to inhibit S. bovis. A wild type (WT) pure culture of A. migulance strain, FA2B, was grown and used to KO each using the CRISPR-Cas9 system. Resulting data identified 40 FA2B KOs for Bacteriocin1. All KO isolates picked were unable to inhibit S. bovis. However, FA2B KOs for the terpene gene, Bacteriocin 2 & 3, and for the T3PKS gene, were able to inhibit S. bovis, confirming that the gene Bacteriocin1 was the gene responsible for antibacterial activity of A. migulance strain FA2B against S. bovis. To confirm Bacteriocin1 was KO from FA2B in the mutants, PCR assays were used against Bacteriocin1 using gene specific primers. A PCR assay targeting Bacteriocin1 gene sequence confirmed FA2B-Bacteriocin1 KO mutants to no longer carry an active Bacteriocin1 gene. However, the FA2B-WT strain showed a positive PCR result for the Bacteriocin1 gene. Additionally, specific primers designed upstream and downstream of the CRISPR target site were used on both the FA2B-WT and FA2B-Bacteriocin1 mutants to further ensure disruption of Bacteriocin1 gene in the mutants. Random transposon mutagenesis was utilized to insert the identified Bacteriocin1 gene into a GRAS-approved strain of B. pumilus (11E). A total of 2,206 colonies of the B. pumilus (11E) mutants were picked and screened against S. bovis. Out of all the isolates, 122 isolates inhibited S. bovis and contained a functional gene copy of Bacteriocin1 that could inhibit S. bovis. Gene specific primers against Bacteriocin1 gene was used to identify that the gene has been inserted into the B. pumilus (11E) genome. The wild type B. pumilus (11E) was unable to amplify the Bacteriocin1 gene as such amplifying the bacteriocin gene from the mutants after transposon mutagenesis confirms the insertion of the gene to the B. pumilus (11E) strain.

Implications

This is one of the first studies utilizing gene editing techniques to develop novel probiotics for animal agriculture. In future studies this strain can be further modified using pH induced promoters to increase bacteriocin production that would lead to better protection under acidotic conditions and to reduce or control acidosis by responding to environmental cues. At present the location where the Bacteriocin1 gene was inserted in the B. pumilus genome has not yet been identified. Currently, whole genome sequencing is under way to identify the location of insertion. This study is the first step towards developing engineered DFMs to increase animal health and performance. Similar strategies can be used to develop DFMs that respond to environmental cues to increase the therapeutic potential of the microbiome.