Gut check microbiome update

In this issue

Microbiomes further afield—Agricultural applications

The interest and utility of the microbiome is certainly not limited to human medicine. The microbiome has long been of interest in agricultural circles and is of increasing importance particularly as the demand for organic and non-GMO agriculture continues to expand. While issuing patent claims in this area often mirrors the trends seen in medical microbiome applications, the differences can be illuminating and suggest opportunities for those aiming to apply microbiome advances to clinical applications as well as those pursuing IP protection in agricultural areas.

A look at issuing patents:

An ever-present concern in microbiome-focused patent application is the definition of the subject microbes. Regular readers and those otherwise familiar with this area are well aware of the early reliance on 16S rRNA sequences, as well as the more recent trends to use genomic- or transcriptomic-based definitions. Each of these approaches attempts to address the need to define the microbes in a way that the USPTO will consider appropriately definite, without excluding functionally equivalent microbes from patent protection.

Different approaches may be more or less appropriate depending on the particular technological application and the identity of the relevant microbe(s). Additionally, preparing a patent application that includes multiple different approaches to microbe definition is the best way to provide the necessary ability to respond to emerging trends and new developments in this area.

Past successes in patent prosecution are an often fertile ground for how to approach both applications currently in front of the USPTO as well as IP that is yet to be filed upon. The following examples include recently issued claims from the agricultural microbiome space, which are educational to all of those with an interest in microbiome IP trends regardless of their ultimate commercial intentions.

Claims defining microbes by 16S sequence

U.S. Patent 10,271,554

• Titled: Plants containing beneficial endophytes
• Inventors: Mitter, et al.
• Assignees: Indigo Ag, Inc. (Boston, MA), AIT Austrian Institute of Technology Gmbh (Vienna, AT)
• Issued: April 30, 2019

Claim of interest:

• 1. A method for generating an agricultural seed internally colonized with a bacterial endophyte, the method comprising: contacting at least one flower of a plurality of agricultural plants with a synthetic formulation comprising a purified bacterial population, the bacterial population comprising a bacterial endophyte in an amount effective to internally colonize at least one seed made by the flower, wherein the bacterial endophyte exhibits the ability to produce cellulase activity and pectinase activity, wherein the contacting results in a concentration of at least 10 CFU of the bacterial endophyte inside the tissue of the at least one internally colonized seed; and the bacterial endophyte is of the genus Enterobacter and comprises a 16S nucleic acid sequence comprising SEQ ID NO:905.

Multiple tables worth of bacterial endophytes are identified in the specification by name and 16S nucleic acid sequence. The specification also refers to “a bacterial endophyte comprising a 16S nucleic acid sequence at least 97% identical” to the exemplified sequences.

The noted claim defines the bacteria to be used by identity to a single 16S sequence. This limitation was added during prosecution.

Claims defining functional characteristics

U.S. Patent 10,212,941

• Titled: Method for treatment and control of plant disease
• Inventors: Gonzalez, et al.
• Assignees: The Texas A&M University System (College Station, TX)
• Issued: February 26, 2019

Claim of interest:

• 1. A method of preventing or reducing symptoms or disease associated with Xanthomonas axonopodis pv. citri or Xylella fastidiosa in a plant, comprising contacting said plant with a population of virulent bacteriophage particles that includes Xylella fastidiosa in its host range, wherein the virulent bacteriophage is selected from the group consisting of:
  
  the Xfas100 phage type and the Xfas300 phage type;
  
  wherein the Xfas100 type phage has the following characteristics:

1. the bacteriophage is capable of lysing said Xylella fastidiosa and/or Xanthomonas bacteria;
2. the bacteriophage infects a cell by binding to a Type IV pilus;
3. the phage belongs to Siphoviridae family and has capsid size ranging from 55-77 nm in diameter;
4. the genomic size of bacteriophage is about 55500 bp to 56200 bp; and
5. the bacteriophage prevents or reduces symptoms associated with X. fastidiosa or Xanthomonas axonopodis pv. citri and subspecies thereof, in a plant or plants; and

wherein the Xfas300 type phage has the following characteristics:

1. the bacteriophage is capable of lysing said Xylella fastidiosa and/or Xanthomonas bacteria;
2. the bacteriophage infects a cell by binding to a Type IV pilus;
3. the phage belongs to Podoviridae family and has capsid size ranging from 58-68 nm in diameter;
4. the genomic size of bacteriophage is about 43300 bp to 44600 bp; and
5. the bacteriophage prevents or reduces symptoms associated with X. fastidiosa or Xanthomonas axonopodis pv. citri and subspecies thereof, in a plant or plants.

This method claim describes phage by broad genus/family classifications and a combination of a number of structural and functional characteristics. The claim lacks any direct sequence limitations. A dependent claims lists specific phage types/Accession Numbers.

Claims defining microbial combinations and 16S sequence

U.S. Patent 10,212,944

• Titled: Designed complex endophyte compositions and methods for improved plant traits
• Inventors: Ambrose, et al.
• Assignees: Indigo Ag, Inc. (Boston, MA)
• Issued: February 26, 2019

Claim of interest:

• 1. A synthetic composition comprising a designed complex endophyte and an agronomic formulation, the designed complex endophyte comprising a host fungal endophyte comprising a heterologous bacterial endophyte living inside the host fungal hyphae, wherein the designed complex endophyte provides a trait of agronomic importance to an agricultural plant element to which the synthetic composition is applied or to an agricultural plant grown from said agricultural plant element or of which said agricultural plant element is a part.

The claim describes a combination of a fungal endophyte and bacterial endophyte, where the combination is heterologous. Dependent claims list multiple suitable genera for each endophyte, as well as describing suitable endophytes by 95% identity to any of over 500 16S or ITS RNA sequences.

Claims defining microbial combinations

U.S. Patent 10,154,671

• Titled: Bacterial composition for improving plant growth
• Inventors: Cor et al.
• Assignees: Lellemand Plant Care SAS
• Issued: December 18, 2019

Claim of interest:

• 1. A composition for improving plant growth, comprising at least one bacterium live strain of the Lactobacillus rhamnosus species, in combination with at least one inactivated or extract fungi and/or yeast, wherein Lactobacillus rhamnosus is the only Lactobacillus species present in the composition and wherein the at least one bacterium live strain of the Lactobacillus rhamnosus species improves plant growth over an untreated plant with or without exposure to a pathogen.

This composition claim combines a partly isolated species with fungi/yeast material and functional limitations. A dependent claim provides a deposit number for a preferred strain.

U.S. Patent 10,051,869

• Titled: Compositions and methods for improving plant health
• Inventors: Smith et al.
• Assignees: HSC Organics (Apollo Beach, FL)
• Issued: August 21, 2019

Claim of interest:

• 1. A composition for improving plant health, the composition comprising a synergistic ratio of (i) composting bacteria, (ii) bio-surfactant producing bacteria, (iii) toxin remediating bacteria, (iv) bovine feces, and (v) humic acid,
  
  wherein the composting bacteria comprise at least three of Nocardioides kongjuensis, Clostridium phytofermentans, Clostridium cellulolyticum, Comamonas jiangduensis, and Bacillus subtilis,
  
  the bio-surfactant producing bacteria comprise at least one of Comamonas jiangduensis and Pseudomonas aeruginosa, and
  
  the toxin remediating bacteria comprise at least one of Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas fluorescens, and Comamonas testosteroni, and
  
  wherein the synergistic ratio is approximately between 10⁴ to 10¹² colony-forming units (CFU) per ml for each bacterial species, approximately between 1% and 25% (w/v) bovine feces, and approximately between 0.01% and 1.00% (w/v) humic acid.

This composition claim combines multiple species-limited microbes at certain concentrations, combined with fertilizer ingredients.

Conclusions

Before turning to the issue of defining microbes in a claim, it is noted that the examples above highlight the power of the term “heterologous” to distinguish compositions from natural products. Such language is a direct outcome of the Myriad decision on patent-eligible subject matter and may eventually become as ubiquitious as “isolated” was in pre-Myriad patents.

Agricultural microbiome claims continue to issue that define microbes by classic, widespread approaches such as 16S or ITS sequences. We note that the above examples may indicate that claims are more likely to be permitted to recite a large number of individual sequences when the 16S sequence is not featured in the independent claim—lessening the need for a long sequence of continuations and/or divisionals while still providing the advantages of a sequence claim.

Another standby approach in microbiome IP is defining the microbe by functional descriptions. While, in recent years, functional limitations have come under more critical review at the USPTO (a trend that extends well beyond the microbiome space) and purely functional limitations can be difficult to obtain, it is clear from the above that such an approach can still succeed, particularly when combined with some structural descriptions.

A currently popular trend in the agricultural microbiome space that is of particular interest is the use of combination of microbes (usually described at the species or genus level, although even broader claims are possible) with a second microbe and/or additional elements of a formulation. This approach can provide striking scope in the identity of the individual microbes if the applicant is able to include limitations that further describe their commercial formulation’s other aspects. Prior issues of Gut Check have reviewed the use of such combinatorial approaches in method of treatment claims, but this approach can see success for composition claims as well.

As ever, the savvy applicant will incorporate as many different approaches as is practicable, and the patents above each exemplify a defense in depth approach—multiple different approaches to microbe definition and/or inclusion of deposit or accession-specific dependent claims. Such forward-thinking approaches provide claim sets that will be more likely to weather shifting trends and unexpected court rulings.