DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/15/2025 has been entered.
Status of Claims
Receipt of Arguments/Remarks filed on 12/15/2025 is acknowledged. Claims 1-30 are pending. Claims 1, 2, 10, 20, 21, 29, and 30 were amended. Claims 12-16, 18, and 19 have been withdrawn from consideration. Claims 1-11, 17, and 20-30 are currently under consideration.
Information Disclosure Statement
The information disclosure statement filed 12/16/2025 fails to comply with 37 CFR 1.98(a)(3)(i) because it does not include a concise explanation of the relevance, as it is presently understood by the individual designated in 37 CFR 1.56(c) most knowledgeable about the content of the information, of each reference listed that is not in the English language. The references not considered by the Examiner are lined through on the IDS form.
Claim Objections
Claim 29 is objected to because of the following informalities: in line 2 of claim 29, there should be a comma between “Snodgrassella alvi” and “Lactobacillus spp.” Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 4, 5, 24, and 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 4, 5, 24, and 25 recite “103 viable bacteria cells” and “106 viable bacterial cells”. It appears that 103 should instead be 103, and 106 should instead be 106. However, as currently written this is not clear, rendering the scope of these claims indefinite. Appropriate correction is required.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 20, 22-23, and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Ludvigsen et al., Microbes and environments; 30(3):235-44 in view of Horton et al., PeerJ; 3:e1329, and as evidenced by Kwong WK et al., Nature reviews microbiology; 14(6):374-84 (hereinafter referred to as “Kwong WK”).
Regarding claims 1 and 29, Ludvigsen teaches that the gut microbiome composition of honey bees comprises Snodgrassella spp., Lactobacillus spp., Gilliamella spp., and Bifidobacterium spp. (Ludvigsen Fig. 1; Table 2). Ludvigsen teaches a co-culture comprising Snodgrassella alvi and Gilliamella apicola (Ludvigsen Fig. 4; pp. 237-238 “Competition experiment”).
“Aerobic” as recited in claim 1 is not specifically defined in the disclosure, and under broadest reasonable interpretation an aerobic co-culture reads on a co-culture wherein any amount of oxygen is present. Ludvigsen teaches that the bacteria are grown together in tryptic soy broth (TSB) in 1.5-mL Eppendorf tubes prepared with acid-washed glass beads to obtain the co-culture (Ludvigsen p. 238 “Competition experiment”). Ludvigsen does not teach that the co-culture conditions require any step or apparatus for completely removing oxygen from the environment or creating an anaerobic environment, and thus it is considered that this co-culture composition comprises some amount of oxygen and is therefore aerobic.
Regarding claims 20 and 22, Ludvigsen teaches that the bacteria are in a liquid growth medium, tryptic soy broth, and thus teaches an aerobic bacterial growth medium comprising Snodgrassella alvi and Gilliamella (Ludvigsen p. 238 first partial para.).
Ludvigsen does not teach a co-culture or growth medium comprising Snodgrassella alvi and Lactobacillus spp. in addition to at least one other claimed bacterial strain (claims 1, 20, 22-23, 29).
Regarding claims 1 and 29, Horton teaches that three major bacterial phyla dominate the honey bee microbiome and include the Proteobacteria (including Gilliamella, Frischella, and Parasaccharibacter) the Firmicutes (e.g., Lactobacillus sp. Firm-4 and Firm-5), and the Actinobacteria (such as Bifidobacterium) (Horton p. 2 first partial para.). Horton teaches that core honey bee microbiome members include Snodgrassella species, Lactobacillus firm-5 and firm-4, alpha-2.1, and alpha-2.2 (Horton p. 5 “Foraging bees from both productive and unproductive colonies host many bacterial genera”). Horton teaches that there is interest in studying the benefits of feeding probiotic bacteria to bee colonies, which requires studying the gut microbial communities in honeybees, and that the microbial communities in the bee gut contribute to the health of the bee (Horton p. 1 para. 1, p. 2 first partial para.). Horton teaches co-cultures of a variety of honey bee microbiome isolates, including Lactobacillus and Gilliamella (Horton p. 4 “Co-culture and microbiological protocols”; Fig. 4).
Regarding claims 22 and 23, Horton teaches co-cultures in liquid medium and on solid nutrient agar plates (Horton Fig. 4).
It would have been obvious to a skilled artisan, before the effective filing date, to obtain a co-culture or co-culture growth medium comprising Snodgrassella alvi, Lactobacillus, and Gilliamella. Ludvigsen teaches a co-culture of Snodgrassella alvi and Gilliamella, and Horton teaches a co-culture comprised of Lactobacillus and Gilliamella. Further, both of these references teach that Snodgrassella alvi, Lactobacillus, and Gilliamella are all commonly present as core members of the honey bee gut microbiome. For these reasons, a skilled artisan would have found it obvious to modify the teachings of Ludvigsen and obtain a co-culture and medium that contains Lactobacillus in addition to Snodgrassella alvi and Gilliamella.
A person having ordinary skill in the art would have been motivated, with a reasonable expectation of success, to obtain a co-culture comprising Snodgrassella alvi, Lactobacillus, and Gilliamella, given the presence of all of these microorganisms as part of the honey bee gut microbiome. As the gut microbial community is known to influence the health of bees through a variety of factors such as the expression of various enzymes and pathways by the gut bacteria (Horton p. 2 first partial para.), characterizing the interactions of these microbes and understanding how they influence each other and in turn the health and fitness of the honey bee is of major interest. Thus, a skilled artisan would have been motivated to obtain co-cultures of a variety of microbes that colonize the honey bee gut to better understand this environment and how the microbes are interacting. This is especially useful in understanding the role of bee gut microbes for the purpose of developing probiotics to improve the health and fitness of bee colonies.
There would have been a reasonable expectation of success in obtaining a co-culture comprising Snodgrassella alvi, Lactobacillus, and Gilliamella, as co-cultures of Gilliamella with Snodgrassella alvi or Gilliamella with Lactobacillus are both known in the art, and therefore an ordinary artisan could expect success in obtaining a co-culture of all three of these microorganisms together, all of which are found naturally in the honey bee gut.
Regarding the recitation of “aerobic” in claims 1 and 20, as discussed above, it appears that Ludvigsen teaches an aerobic co-culture comprising Snodgrassella alvi and Gilliamella, as there are no steps of the culturing technique indicating that oxygen has been removed. Further, it is known in the art that S. alvi relies on aerobic respiration (see Kwong WK p. 9 para. 2) and that the microorganisms found in the honey bee gut, including Gilliamella and Lactobacillus, are facultative anaerobes or microaerophiles, indicating that they are capable of growth in the presence of oxygen (see Kwong WK p. 4 para. 2). Therefore, especially given the requirement of S. alvi for aerobic respiration, it would have been obvious for a skilled artisan to obtain an aerobic co-culture of S. alvi, Lactobacillus, and Gilliamella, wherein oxygen is present.
Regarding the recitation of “stable” in claims 1, 20, and 29, “stable” is not defined in the instant specification to indicate what parameters are used to determine a stable co-culture, the threshold of stability that is required to be considered stable, or the structure of a stable co-culture versus an unstable co-culture. As such, under broadest reasonable interpretation, a stable co-culture reads on any composition in which two or more microorganisms have been grown together in the same conditions and are present together simultaneously in a culture medium. Thus, the co-cultures taught by Ludvigsen and Horton are both considered to be stable co-cultures.
Regarding the “wherein” clauses of claims 1 and 29, these are product-by-process limitations, and patentability is assessed based on the structure implied by the steps, not the manipulations of the recited steps. However, Ludvigsen teaches that the co-culture is obtained by propagating the bacteria together as a single culture, or that they were grown together (Ludvigsen p. 238 first partial para.). Horton similarly teaches that the various honey bee microbiota strains, including Lactobacillus and Gilliamella, are propagated together in co-culture (Horton p. 5 first partial para.).
Claims 2, 21, and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Ludvigsen and Horton as applied to claims 1, 20, 22-23, and 29 above, and further in view of Kwong et al., Genome announcements; 2(6):10-128; and as evidenced by NCBI GenBank NR_122055 and NR_121727.
Regarding claims 2, 21, and 30, Ludvigsen and Horton teach the co-culture composition of claims 1 and 29. Ludvigsen teaches that the Snodgrassella alvi strain has GenBank Accession Number NR_122055, and that Gilliamella apicola has GenBank Accession Number NR_121727 (Ludvigsen Table 2). These GenBank Accession Numbers correspond with the Snodgrassella alvi wkB2 16S ribosomal RNA sequence and Gilliamella apicola strain wkB1 16S ribosomal RNA (see NCBI GenBank NR_122055 and NR_121727). Thus, Ludvigsen teaches a co-culture comprising Snodgrassella alvi wkB2 and Gilliamella apicola strain wkB1. Horton teaches that Lactobacillus Firm-5 is a core honey bee microbiome member (Horton p. 5 “Foraging bees from both productive and unproductive colonies host many bacterial genera”).
These references do not teach that the Lactobacillus strain is Lactobacillus "Firm-5" wkB10 or wkb8 as set forth in claims 2, 21, or 30.
Regarding claims 2, 21, and 30, Kwong teaches the genome sequences of Lactobacillus sp. strains wkB8 and wkB10, members of the Firm-5 Clade, from honey bee guts (Kwong “Abstract”). These strains were isolated from the honey bee gut and Firm-5 strains are commonly identified as the most numerically abundant phylotype in honey bee guts (Kwong p. 1 para. 1-2).
It would have been obvious for a skilled artisan to utilize the specific Lactobacillus sp. strains Firm-5 wkB8 and wkB10 as taught by Kwong in a co-culture composition as taught by Ludvigsen and Horton. All of these references teach that Lactobacillus sp. are predominant members of the honey bee gut microbiome. Horton and Kwong teach that Firm-5 strains are highly abundant in the honey bee gut environment. Thus, a skilled artisan would have found it obvious to utilize a Firm-5 strain that was isolated from the honey bee gut, wkB8 or wkB10, in a co-culture composition comprising other honey bee gut microbes such as Snodgrassella alvi or Gilliamella apicola.
A person of ordinary skill in the art would have been motivated to utilize the strains taught by Kwong in a co-culture composition comprising bacterial strains from honey bees such as Snodgrassella alvi or Gilliamella apicola because wkB8 and wkB10 are both highly abundant in the gut microbiome of honey bees, and it would therefore be of interest to characterize interactions of these microbes with other members of the gut microbiome. As taught by Horton, it is of significant interest to characterize the gut microbial community of honey bees as these microbes influence the health of the bees, and in turn influences agriculture and honey production (Horton “Introduction”).
A skilled artisan would have had a reasonable expectation of success in obtaining a co-culture with Snodgrassella alvi wkB2, Gilliamella apicola wkB1, and Lactobacillus Firm-5 wkB8 or wkB10 because all of these strains are known to exist naturally in the honey bee gut as taught by Ludvigsen and Horton, and co-cultures including other Lactobacillus strains from the honey bee gut microbiome have been successfully obtained as taught by Horton.
Claims 3-5, 7-9, 11, 17, 24-25, and 27-28 are rejected under 35 U.S.C. 103 as being unpatentable over Ludvigsen and Horton as applied to claims 1, 20, 22-23, and 29 above, and further in view of Olofsson US 2017/0226599 A1.
Ludvigsen and Horton teach the compositions of claims 1 and 20 as set forth above. These references do not teach the effective amount as recited in claim 3, bacterial cells per gram recited in claims 4-5 and 24-25, the lyophilized form of bacterial strains recited in claims 7 and 27, the antibiotic in claims 8 and 28, the ingestible composition recited in claims 9 and 11, or the article of manufacture of claim 17.
Regarding claim 3, the instant specification, p. 10, states that an “effective amount” is that amount of a compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. Olofsson teaches Lactobacillus strains isolated from honeybees and teaches that these bacterial strains are used in medically related products such as food, beverage, feed and wound care. (Olofsson p. 1 para. 1). Olofsson teaches probiotic feed products for bee and bee larvae comprising an effective amount of the bacterial strains from the honeybee gut to strengthen or re-establish the microbial flora within the bee or bee larvae and re-establish the inhibitory atmosphere inside the beehive (Olofsson p. 5 para. 67). Thus, Olofsson teaches a composition comprising an effective amount of honey bee gut bacteria.
Regarding claims 4-5 and 24-25, Olofsson teaches a feed product for administration to honey bees comprising the bacterial strains at a concentration from about 101 to 1014 CFU/g product (Olofsson p. 5 para. 69-70). It is noted that where the claimed ranges "overlap or lie inside the ranges disclosed by the prior art" and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (see MPEP 2144.05 I).
Regarding claims 7 and 27, Olofsson teaches that the feed product may be freeze-dried or lyophilized (Olofsson p. 5 para. 71).
Regarding claims 8 and 28, Olofsson teaches that medical products comprising the bacterial strains may contain antibiotics (Olofsson p. 4 para. 64).
Regarding claims 9 and 11, Olofsson teaches that a composition comprising an effective amount of bacteria is a feed product, i.e. ingestible, and that the ingestible composition contains pollen or sucrose (Olofsson p. 5 para. 72 and 74).
Regarding claim 17, Olofsson teaches that a powdered feed product that is simple to
handle, transport, store, and has a more expanded to date of expiration (Olofsson p. 5 para. 71). Therefore, Olofsson teaches that the composition comprises or is contained in packaging material or stored in a package, i.e., is an article of manufacture.
It would have been obvious to a skilled artisan, before the effective filing date, to modify the teachings of Ludvigsen and Horton, and create a co-culture or growth media composition comprising an effective amount of the honeybee gut bacterial strains, using the concentrations taught by Olofsson. It would have further been obvious to create an ingestible product in a lyophilized form, comprising antibiotics, pollen and sucrose as taught by Olofsson. Packaging such a product as an article of manufacture would also have been obvious, for storage purposes and commercial use. All of these references teach compositions comprising bacteria derived from honey bee guts, and all of the references teach that Lactobacillus are major constituents of the honeybee gut microbiome. It would have been obvious to adapt the teachings of Ludvigsen and Horton to create an ingestible composition or article of manufacture comprising effective amounts of Snodgrassella alvi, Lactobacillus, and Gilliamella co-cultures, based on the teachings of Olofsson directed to such a composition with honeybee-derived Lactobacillus.
A skilled artisan would have been motivated to utilize concentrations in the range taught by Olofsson because these concentrations are effectively used in feed products for honeybees comprising honeybee gut bacteria to achieve beneficial effects. A skilled artisan would have similarly been motivated to include other components of the ingestible composition taught by Olofsson to be useful, such as antibiotics for protection against pathogens in a medical product, or pollen and sucrose to aid in ingestion of the product by the bees. An ordinary artisan would have been motivated to use a lyophilized form of the bacteria because Olofsson teaches that lyophilized or freeze-dried bacterial products can be easily handled, transported, stored, and have expanded expiration dates (Olofsson para. 71). Therefore, it would be considered advantageous to formulate the co-culture compositions taught by Ludvigsen and Horton in lyophilized form. It would similarly be advantageous to create a composition of the co-culture that is packaged as an article of manufacture for extended storage purposes.
A skilled artisan would have reasonable expectation of success in modifying the prior art reference to arrive at the claimed invention given the teachings of Ludvigsen and Horton directed to co-cultures with these bacterial strains and the teachings of Olofsson that honey bee gut-associated bacteria, specifically Lactobacillus spp., can be incorporated into an ingestible and commercial product comprising the components as recited in claims 3-5, 7-9, 11, 17, 24-25, and 27-28.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Ludvigsen, Horton, and Olofsson as applied to claims 3-5, 7-9, 11, 17, 24-25, and 27-28 above, and further in view of Kwong et al., Genome announcements; 2(6):10-128; and as evidenced by NCBI GenBank NR_122055 and NR_121727.
Regarding claim 9, Ludvigsen, Horton, and Olofsson teach the ingestible composition of claim 9 comprising the effective amount of co-culture of claim 1, as set forth above. Ludvigsen teaches Snodgrassella alvi wkB2 and Gilliamella apicola strain wkB1 as discussed above (see NCBI GenBank NR_122055 and NR_121727). Horton teaches that Lactobacillus Firm-5 is a core honey bee microbiome member (Horton p. 5 “Foraging bees from both productive and unproductive colonies host many bacterial genera”).
These references do not teach that the Lactobacillus strain is Lactobacillus "Firm-5" wkB10 or wkb8 as set forth in claim 10.
Regarding claim 10, Kwong teaches the genome sequences of Lactobacillus sp. strains wkB8 and wkB10, members of the Firm-5 Clade, from honey bee guts (Kwong “Abstract”). These strains were isolated from the honey bee gut and Firm-5 strains are commonly identified as the most numerically abundant phylotype in honey bee guts (Kwong p. 1 para. 1-2).
It would have been obvious for a skilled artisan to utilize the specific Lactobacillus sp. strains Firm-5 wkB8 and wkB10 as taught by Kwong in an ingestible composition as taught by Ludvigsen, Horton, and Olofsson. All of these references teach that Lactobacillus sp. are predominant members of the honey bee gut microbiome. Horton and Kwong teach that Firm-5 strains are highly abundant in the honey bee gut environment. Thus, a skilled artisan would have found it obvious to utilize a Firm-5 strain that was isolated from the honey bee gut, wkB8 or wkB10, in an ingestible co-culture composition comprising other honey bee gut microbes such as Snodgrassella alvi or Gilliamella apicola.
A person of ordinary skill in the art would have been motivated to utilize the strains taught by Kwong in an ingestible composition comprising bacterial strains from honey bees such as Snodgrassella alvi or Gilliamella apicola because wkB8 and wkB10 are both highly abundant in the gut microbiome of honey bees, and it would therefore be of interest to include bacteria known to be highly abundant in this environment in probiotic and therapeutic feed products for administration to bees.
A skilled artisan would have had a reasonable expectation of success in obtaining a co-culture with Snodgrassella alvi wkB2, Gilliamella apicola wkB1, and Lactobacillus Firm-5 wkB8 or wkB10 because all of these strains are known to exist naturally in the honey bee gut and co-cultures including other Lactobacillus strains from the honey bee gut microbiome have been successfully obtained as taught by Horton.
Claims 6 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Ludvigsen, Horton, and Olofsson as applied to claims 3-5, 7-9, 11, 17, 24-25, and 27-28 above, and further in view of Le, US 2018/0192668 A1.
Ludvigsen, Horton, and Olofsson teach the compositions of claims 1, 3, and 20 as set forth above. These references do not teach the sporulated microbial strains of claims 6 or 26.
Regarding claims 6 and 26, Le teaches a method of making and using an animal feed containing strains of sporulated living microbes to stimulate local intestinal immunity with the microbes (Le Abstract). Le teaches selecting a plurality of strains of sporulated living microbes to form a microbial solution, wherein the ability to form spores allows germination in the upper digestive tract for nutrient absorption and stimulate local intestinal immunity (Le claim 1, para. 21 and 28).
It would have been obvious to a skilled artisan, before the effective filing date, to modify the composition of Ludvigsen, Horton, and Olofsson to utilize sporulated forms of the microbes as taught by Le, as these references all relate to bacterial compositions for beneficial alterations to the gut microbiota composition.
A skilled artisan would have been motivated to combine these teachings because Le
teaches that the sporulated bacterial strains display activity in the upper digestive tract helping
with nutrient absorption and stimulate local intestinal immunity (Le para. 28). Therefore, it would be considered advantageous to incorporate a sporulated formulation in the composition of Ludvigsen, Horton, and Olofsson.
The ordinary artisan would have reasonable expectation of success in combining these teachings to obtain a co-culture composition as taught by Ludvigsen, Horton, and Olofsson comprising a sporulated bacterial strain as taught by Le, given the successful use of sporulated bacterial strains by Le in compositions to improve the gut microbiome.
Response to Arguments
In light of amendments to the claims, the rejections under 35 U.S.C. § 103 as set forth in the previous Office Action been withdrawn. However, upon further consideration, new grounds of rejection of the pending claims are made as set forth above. Given these new grounds of rejection and new prior art references applied, the arguments presented regarding claims rejected under 35 U.S.C. § 103 as they pertain to prior art teachings are moot.
Regarding the arguments directed to surprising and unexpected results, specifically that the claimed product produced by co-culturing the claimed bacterial strains as a single culture prior to administration to the bee gut increases stability and uniformity of the probiotic inoculation, and that the claims have been amended to recite unexpected features not present in the prior art (stable co-culture), these arguments are not persuasive.
In response to these arguments, it is noted that the present invention is directed to a co-culture or growth medium composition comprising two or more of the claimed microbes associated with the bee microbiome. The experimental results referred to by applicant, Figures 1 and 2 of the instant application, are directed to the stability and uniformity of the bee gut microbiome composition after inoculation with the co-culture compositions. These experimental results, or any part of the instant disclosure, does not teach or indicate that the co-culture products, to which the instant claims are directed, have any structural difference in stability or uniformity due to the production method set forth in the product-by-process limitations of claims 1 and 29. The results regarding stability and uniformity refer only to the composition of the bee gut microbiome. Additionally, these results compare a co-culture inoculation of bees to a single culture inoculation, showing that the gut microbiome of the bees differs when administered a co-culture compared to an individual culture. However, the prior art teaches co-cultures of these microbes. Thus, the evidence presented that a co-culture produces different results than a single culture does not differentiate the instant invention from the prior art teachings.
Further, as discussed above, the disclosure does not define a stable co-culture, and it is considered that the prior art references cited teach stable co-cultures, wherein multiple bacterial strains are present simultaneously in a final co-culture product. Thus, it is not considered that the claimed co-culture products possess unexpected properties to differentiate from the prior art teachings, which are directed to co-culture compositions comprising the claimed strains as discussed above.
Conclusion
Claims 1-11, 17, and 20-30 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653