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 .
DETAILED ACTION
Claims 6, 15, and 21 are canceled. Claims 24-25 are new. Claims 1-5, 7-14, 16-20 and 22-25 are pending and under consideration.
Priority
The claims are entitled to the effective filing date of 04/06/2020.
Claim Rejections - 35 USC § 112(b)
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 1-5, 7-14, 16-20 and 22-25 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.
Claim 1, as amended, recites the limitation “the analytical sample” in line 19. There is insufficient antecedent basis for this limitation in the claim. It is uncertain to which of the two earlier recited analytical samples the “analytical sample” limitation in line 19 was making reference. Claim 1 recites a treated analytical sample(s) and a control analytical sample(s) in lines 12-13. As such, it is unclear whether “the analytical sample” recited in line 19 refers to the treated analytical sample(s) or the control analytical sample(s).
Claim 1, as amended, further recites the limitation “so as to concentrate species of the analytical sample” in lines 18-19 and the limitation “the concentrated species” in line 21. There is insufficient antecedent basis for these limitations in the claim. Since there is no earlier requirement in claim 1 for an analytical sample with a species, it is unclear which species is required to be concentrated in lines 18-19. As such, it is unclear whether the “species of the analytical sample” recited in lines 18-19 and “the concentrated species” recited in line 21 refer to the treated portion of each microbial population or the untreated portion of each microbial population.
Claims 2-5, 7-14, 16-20 and 22-25 depend from claim 1 and are rejected for the reason set forth above.
(Maintained) Claim 13 recites the phrase “in particular” in line 3. This phrase renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. Claim 13 recites “Robs = trn/t0 or any other method of determination” in the last two lines, which renders the claim indefinite because the scope encompassed by “any other method of determination” is unclear.
Claim 17, as amended, is drawn to a kit for carrying out the process of claim 1. Claim 17 recites the limitation “the analyzed bacterial population” in lines 7-8. There is insufficient antecedent basis for this limitation in the claim. Furthermore, claim 17 is missing a conjunction in front of the last listed component, i.e. in front of “the at least one separation channel”. It is unclear whether the claim is incomplete or whether both of the listed limitations are required because there is no conjunction between the listed items. As such, one of ordinary skill in the art cannot ascertain the metes and bounds of the kit required in claim 17.
Claim 23 depends from claim 17 and is rejected for the reason set forth above.
New claim 25 is indefinite because there is no period and, therefore, it is unclear whether the claim is complete. MPEP 608.01(m) states that [e]ach claim begins with a capital letter and ends with a period. Furthermore, claim 25 recites the limitation “the species of the microbial population” in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 25 depends from claim 1, which does not require a species of microbial population.
Response to Arguments
Applicant's arguments filed 07/10/2026 have been fully considered to the extent that they might apply to the new grounds of rejection set forth above, but they are unpersuasive.
Rejection of claims 1-5, 7-14, 16-20 and 22-25 under 35 U.S.C. 112(b)
Applicant argues that to the extent any recitation of claim 13 remains at issue, Applicant asserts that claim 13 is definite when read in light of the specification (Spec. p. 10, ll.7-14). See the remarks p. 11 para 2.
This argument is not persuasive because page 10 lines 7-14 of the specification filed 09/15/2025 states “Furthermore, the signal(s) contained in the elution profile(s) of the analytical sample(s) which are analyzed with regard to the variability thereof are namely the peak position defined by the t-r measured at peak apex, or defined by the peak median, or normalized by the calculation of the retention factor, Robs= trn/t-0, or any other method of determination, or the peak width of the elution profile. The variation of the signals contained in the elution profiles will then be quantified.” This section of the specification does not clarify the metes and bounds encompassed by “any other method of determination”, nor does it clarify whether the limitations recited after the phrase “in particular” (line 3) are required in claim 13.
Applicant argues that claims 17 have been amended to depend from claim 1, to recite carrying out the process of claim 1, and the kit recitations have been conformed accordingly, resolving the antecedent basis and conjunction issues previously identified. See the remarks p. 11 para 2.
This argument is not persuasive because the amendment to claim 17 necessitated new grounds of rejection. Claim 17, as amended, depends from claim 1 but claim 1 does not require any bacteria. Rather, claim 1 requires a microbial population. Furthermore, claim 17 was not amended to recite a conjugation in front of the last listed component, i.e. in front of “the at least one separation channel. Therefore, it is still unclear whether claim 17 is complete.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 11-13 and 16 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 11 recites a broader scope of the field-flow fractionation device than that required in claim 1, from which claim 11 depends. Claim 1, as amended, requires a sedimentation field-flow fractionation device to which a multigravitational field is applied (line 15). Claim 11 recites wherein the field-flow fractionation device is a multigravitational or centrifugal, hydrodynamic, dielectrophoretic (DEP), electric, magnetic, thermal field-flow fractionation device. As such, claim 11 is of improper dependent form for failing to further limit the subject matter of the claim upon which it depends.
Claim 12 fails to further limit claim 1 from which it depends. Claim 1, as amended, requires a sedimentation field-flow fractionation device to which a multigravitational field is applied (line 15). Claim 12 recites wherein the field-flow fractionation device is a multigravitational field-flow fractionation device. Since claim 1 requires a multigravitational field to be applied to the sedimentation field-flow fractionation device, the field flow fractionation device is a multigravitational field-flow fractionation device. As such, claim 12 does not further limit the required device of claim 1.
Claim 13 fails to include all limitations of claim 1, from which it depends. Amended claim 1 requires, in lines 29-33, calculating, for each of the treated analytical sample(s) and the control analytical sample(s), a retention factor Robs=trn/t0, where t0 is a dead time of the field-flow fractionation device and calculating a percentage of variation in the retention factor between the treated analytical sample(s) and the control analytical sample(s). Claim 13 recites a broad limitation, wherein the signal or signals contained in the elution profile of the analytical sample(s) which are analyzed with regard to the variability thereof, are in particular the peak position defined by a tr measured at peak apex, or defined by a peak median, or normalized by a calculation of a retention factor, Robs = trn/t0 or any other method of determination, or a peak width of the elution profile. Therefore, claim 13 does not require the Robs=trn/t0 calculation of claim 1, and consequently fails to include all the limitations of the claim upon which it depends.
Claim 16 is drawn to a kit for carrying out the process of claim 1. Claim 1 requires, in lines 15-16, a separation channel of a sedimentation field-flow fractionation device to which a multigravitational field is applied (i.e. external field). Claim 16 recites a broad limitation, at least one separation channel being a flow channel configured for an external field of variable nature to be applied thereon depending on the type of FFF. Therefore, claim 16 fails to further limit the multigravitational sedimentation field-flow fractionation device limitation of claim 1, because claim 16 encompasses any type of field-flow fractionation device and any type of external field of variable nature.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-5, 7-14, 18-20 and 24-25 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more. Each step described below is in reference to the subject matter eligibility test for products and processes (MPEP 2106).
Claim 1 recites “[a] process” which is one of the statutory categories (Step 1: Yes).
Claim 1 requires a step consisting of “quantifying a percentage of variation of the signals contained in the elution profiles by calculating, for each of the treated analytical sample(s) and the control analytical sample(s), a retention factor Robs=trn/t0, where t0 is a dead time of the field-flow fractionation device and calculating a percentage of variation in the retention factor between the treated analytical sample(s) and the control analytical sample(s), and comparing the calculated percentage of variation to a significance threshold, the significance threshold being defined for the microorganisms and the at least one antimicrobial as a function of an active dose of the at least one antimicrobial”. The terms “quantifying” and “comparing” are acts of evaluating information that can be practically performed in the human mind. The specification (filed 10/05/2022) discloses that the variation of the signals contained in the elution profiles is expressed in % and corresponds to a percentage of variation in Robs =PΔR; signifying a biological effect, i.e. the percentage of the variation in the retention factor [Math 1] PΔR= (| Robs | / Robs control)x 100. See page 10 lines 25-31. Furthermore, the instant specification discloses that the “significance threshold” to which the signal variations are compared, is the threshold from which the microbial population is considered to be sensitive or resistant to the antimicrobial. See page 10 line 32 to page 11 line 1. Thus, the instantly claimed quantification and comparison step recited in claim 1 and required by dependent claims 2-14, 18-20 and 24-25 is an abstract idea in the “mental process” judicial exception grouping (MPEP 2106.04(a)(2)(III)) (Step 2A Prong 1: Yes).
Besides the judicial exception, claim 1 recites “[a] process for determining antimicrobial susceptibility of a microorganism in a microbial population from a biological sample to at least one antimicrobial” in lines 1-6. This recitation does not meaningfully limit the claim, because the recitation does not structurally limit the method, and because the recitation merely serves to generally link the judicial exception to a particular technological environment. Before the quantification and comparison abstract idea step, claim 1 requires: “providing at least one microbial population from a biological sample; treating a portion of each microbial population with the at least one antimicrobial, the other portion being untreated with the at least one antimicrobial to serve as a control; incubating the at least one microbial population either with or without the at least one antimicrobial, thereby obtaining the treated analytical sample(s) and the control analytical sample(s), respectively; eluting the treated analytical sample(s) and the control analytical sample(s) from the previous step in a separation channel of a sedimentation field-flow fractionation device to which a multigravitational field is applied, wherein said eluting comprises a stop-flow step in which flow of a mobile phase through the separation channel is interrupted so as to concentrate species of the analytical sample against an accumulation wall of the separation channel under the multigravitational field, and the flow of the mobile phase is thereafter resumed to elute the concentrated species; obtaining via a UV-Vis detector of the field-flow fraction device, elution profiles of the treated analytical sample(s) and the control analytical sample(s) for each microbial population, each elution profile comprising a peak corresponding to the microbial population eluting at a retention time trn”. These pre-solution activities are all data gathering steps, because the purpose of each step is to obtain the information necessary for the abstract idea. Although the steps require the use of a “sedimentation field-flow fractionation device”, this element is merely a tool used to obtain the concept of an elution profile for the purpose of performing the abstract idea. After the quantification and comparison step (abstract idea), claim 1 requires “determining that the microorganism of the microbial population is susceptible to the at least one antimicrobial when the calculated percentage of variation is greater than significance threshold”. This post-solution activity is insignificant, because the step is merely an instruction to apply the judicial exception. Thus, the additional elements of claim 1 do not integrate the judicial exception into a practical application.
Claims 2-5, 7-14, 18-20 and 24-25 depend from claim 1, but do not significantly limit the judicial exception as to integrate it into a practical application. Claim 2 requires the microorganism to be one selected from bacteria, fungi, yeasts and protozoa. Claims 3 and 18 require the at least one antimicrobial to be one selected from antibiotics, antifungals and antiparasitic agents. Claims 4 and 19-20 require the microorganism to be a bacterium. Claim 5 requires the at least one antimicrobial to be an antibiotic. Claim 7 requires the biological sample to be from a human, animal, or environment. Claim 8 requires the biological sample to be urine, stool, sputum, broncho-alveolar water, pus, cerebrospinal fluid, or blood. Claim 9 requires the biological sample to comprise a reference strain. Claim 10 requires the incubation step to last 30-120 minutes. Claims 2-5, 7-10 and 18-20 do not integrate the abstract idea into a practical application because the limitations merely serve to link the abstract idea to the technical field of determining antibiotic resistance in biological samples. Claims 11-12 entail a multigravitational field-flow fraction device. Such element does not integrate the abstract idea into a practical application because the device is merely a tool for obtaining the concept of an elution profile for the purpose of performing the abstract idea. Claim 13 limits the signal or signals contained in the elution profiles. However, the signals encompass mathematical values obtained by observing an output graph from a field-flow fractionation device, such as a tr (i.e. retention time) measured at a peak in the graph. Thus, claim 13 does not add any element to the abstract idea, because claim 13 merely indicates that the quantified and compared signals are obtained by observing an output of a field-flow fractionation device. Claim 14 requires defining a significance threshold for each microorganism/antimicrobial pair. The way in which the significance threshold is to be defined is recited with a high level of generality, such that the step encompasses mentally defining a significant threshold. Claim 24 requires the multigravitational field to be applied at a field strength from 10 to 50 g, and wherein the stop-flow step, the flow of the mobile phase through the separation channel is interrupted for a stop-flow duration sufficient to concentrate the species of the analytical sample against the accumulation wall under the multigravitational field. The limitations of claim 24 do not integrate the judicial exception into a practical application because the limitations are merely instructions for applying the abstract idea. Claim 25 requires the species of the microbial population to have a size of less than 2 µm. The limitation of claim 25 does not integrate the judicial exception into a practical application because the limitation is merely an attempt to generally link the abstract idea to its field of use. Thus, the additional limitations of claims 1-5, 7-14, 18-20 and 24-25 do not integrate the abstract idea into a practical application (Step 2A Prong 2: No).
The additional elements fail to amount to an inventive concept in view of the routine, well understood and conventional activities in the art of determining antibiotic resistance in biological samples. For example, Saenton (Separation Science and Technology, 2000, 35(11), as provided with the IDS filed 10/05/2022), teaches using field flow fractionation to separate living P. putida cells from dead P. putida cells that died in response to an 1% ethanol aqueous solution containing 0.15% 2-methylthiazalone and 0.15% bromo-nitro-dioxane (e.g. a treated analytic sample). See the paragraph spanning pages 1765 to 1766 and figure 4(a). Evidentiary reference Cayman (5-bromo-5-nitro-1,3-Dioxane (CAS 30007-47-7). (2025)) discloses that 5-bromo-5-nitro-1,3- Dioxane is an antimicrobial compound effective against Gram-positive and Gram-negative bacteria (e.g. an antibiotic) and fungi including yeast. See the product description of Cayman. Saenton teaches comparing the elution profiles and retention times of live bacteria (e.g. control analytic sample) injected into a FFF channel to their dead counterparts (e.g. treated analytic sample). See the first paragraph on page 1771. Figure 4(a) shows the elution profile of the live and dead P. putida eluted from the sedimentation FFF. Although Saenton does not teach bacteria obtained from a biological sample, Vaz teaches a method for detecting in a biological sample comprising microorganisms the presence of microorganisms resistant to a predetermined therapeutic agent comprising the following steps: a. inoculating the sample on a first tube with and on a second tube without at least one such therapeutic agent and incubating it. See claim 1. Thus, the instantly claimed field-flow fractionation element was well-known in the art prior to the effective filing date (Step 2B: No).
For all these reasons, claims 1-5, 7-14, 18-20 and 24-25 are not patent eligible.
Response to Arguments
Applicant's arguments filed 07/10/2026 have been fully considered but they are not persuasive.
§ 101 rejection of claims 1-5, 7-14, 18-20 and 24-25
Applicant argues that claim 1 is not directed to the concept of the retention-factor percentage calculation. The specificity of the recited ordered steps integrates any such concept into a practical application and constitutes significantly more, namely, an FFF-based physical separation and antimicrobial-susceptibility determination that cannot reasonably be performed in the human mind and that improves the technical field of microbiological diagnosis. See the remarks p. 12 para. 1.
This argument is not persuasive because the recited steps in claim 1 are either data gathering steps or instructions for applying the judicial exception, as set forth above.
Applicant argues that under step 2A prong 1, claim 1 is not properly characterized as a mental process. Applicant asserts that the claim considered as a whole is not a mental process. See the remarks p. 13 para. 1. Applicant asserts that claim 1 requires physical manipulation and analysis of actual microbial analytical samples. See the remarks p. 13 para. 2. Applicant argues that the limitations in claim 1 cannot be performed in the human mind or with pen and paper. A person cannot mentally elute microbial analytical samples in an FFF device, cannot mentally produce FFF elution profiles or fractograms from treated and control samples, and cannot mentally generate the instrument-produced retention behavior from which the claimed susceptibility determination is made. See the remarks the paragraph bridging pgs. 13-14.
This argument is not persuasive because step 2A prong 1 does not entail analyzing the claim as a whole. Rather, step 2A prong 1 entails determining whether the claim recites a judicial exception including an abstract idea. The “quantifying” and “comparing” steps recited in claim 1 are identified as abstract ideas. Applicant points to the recited steps in claim 1 that require a physical manipulation. However, the additional active method steps that require physical manipulation are not analyzed in step 2A prong 1, because the steps are analyzed as additional elements in step 2A prong 2.
Applicant argues that in step 2A prong 2 claim 1 should not be reduced to the isolated acts of calculating and comparing a retention-factor percentage. The character of the claim as a whole is a physical, instrument-executed microbiological assay in which a treated analytical sample and a control analytical sample are produced, physically eluted in an FFF device, represented as FFF elution profiles, and used to determine whether the microorganism is susceptible to the antimicrobial. See the remarks the paragraph spanning pgs. 14-15. Applicant asserts that claim 1 does not attempt to patent a retention factor percentage or a comparison to a threshold in the abstract. Instead, the calculation is embedded in a physical FFF-based process. See the remarks p. 16 para. 1. The specification explains that SdFFF (sedimentation field flow fractionation) has high sensitivity in terms of changes in size, density, shape, or deformability of analyzed objects, and can be used for early recording of metabolic changes in a cell population without specific sample preparation (spec. p. 4 ll. 10-17). See the remarks p. 16 para. 2. Applicant asserts that the FFF device is not merely a tool in the sense of a generic environment for carrying out an abstract idea. The device performs separation that produces the elution provides. The specification defines an elution profile or fractogram as representing the variation in concentration or quantity of species at the outlet of the separation channel as a function of elution time or volume (spec. p. 9 ll. 13-23). See the remarks the paragraph spanning pgs. 16-17.
This argument is not persuasive because in step 2A prong 2 the claim is considered as a whole. Although the claims require the use of a sedimentation FFF, this particular machine does not integrate the judicial exception into a practical application. MPEP 2106.05(b)(III) states that the “[u]se of a machine that contributes only nominally or insignificantly to the execution of the claimed method (e.g., in a data gathering step or in a field-of-use limitation) would not integrate a judicial exception or provide significantly more.” In the instant case, the claimed sedimentation FFF device is merely used for data gathering. The device is used to obtain elution profiles. In turn, the signals contained in the elution profiles are used for carrying out the “quantifying” and “comparing” steps, i.e. the abstract idea. Therefore, the physical elution in the FFF device does not integrate the judicial exception into a practical application in step 2A prong 2.
Applicant argues that the determining step is not an insignificant post-solution activity. The determining step is the culmination of the claimed physical and analytical procedure. See the remarks p. 17 para. 1. Furthermore, Applicant argues that the end result of the claimed method is a new and useful antimicrobial-susceptibility determination produced b a specific FFF-based laboratory procedure. See the remarks p. 18 para. 1. Applicant asserts that the claim is not the generic use of a known calculation in the environment of microbiology. The specification explains that the inventors’ work led to the first demonstration of the claimed subject matter in the field of microbiology (spec. p. 4, ll. 23-26). See the remarks the paragraph spanning pgs. 18-19. Applicant asserts that the breadth of the recited microorganism and antimicrobial does not make the claim abstract. See the remarks p. 20 last passage.
This argument is not persuasive because the determining step is merely an instruction for applying the results of the abstract idea judicial exception. Applicant asserts that the end result is a new and useful antimicrobial-susceptibility determination. However, the claimed microbial population and the claimed antimicrobial are recited with a high level of generality. MPEP 2106.04(d)(2) states that “[o]ne way to demonstrate such integration is when the additional elements apply or use the recited judicial exception to effect a particular treatment or prophylaxis for a disease or medical condition…If the limitation does not actually provide a treatment or prophylaxis, e.g., it is merely an intended use of the claimed invention or a field of use limitation, then it cannot integrate a judicial exception under the "treatment or prophylaxis" consideration.” In the instant case, the resulting determination of the claimed method does not constitute as a particular treatment or prophylaxis for a disease or medical condition.
Applicant cites the decision of Ex parte Nicula, Appeal 2025-001411 (PTAB Dec. 4, 20205) as an illustrative application of the same framework; the controlling authority remains the Supreme Court and Federal Circuit precedent and the MPEP provisions cited herein. See the remarks p. 21 para. 1. Applicant argues that claim 1 overcomes disadvantages of antimicrobial susceptibility methods currently in use by reciting a specific FFF-based technical process that yields an earlier, instrument-generated susceptibility determination. For these reasons, claim 1 considered as a whole and read in light of the specification integrates the abstract idea into a practical application. See the remarks p. 22 para. 1.
This argument is not persuasive because arguments of counsel cannot take the place of factually supported objective evidence (MPEP 2145 or 716.01(c)). Applicant asserts that the instant method overcomes disadvantages of susceptibility methods currently in use. However, Applicant has not pointed to evidence. Furthermore, the fact pattern set forth in Ex parte Nicula does not match that of the instant claims. On page 17 of the Ex parte Nicula document provided 07/10/2026, it states that “on this record, Appellant’s claimed invention: [P]rovide[s] improved specificity and sensitivity over existing classification techniques”. Applicant asserts that the claim considered as a whole and read in light of the specification integrates the abstract idea into a practical application, but Applicant does not point to evidence of record to substantiate the assertion.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-5, 7, 9-14, 18-20 and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Saenton (Separation Science and Technology, 2000, 35(11), 1761-1775, as previously relied upon) in view of Janča (Journal of Chromatography B, 852(1-2), 512-518, 2007) and Bégaud-Grimaud (Journal of Chromatography A, 1216(52), 9125-9133, 2009) with evidence from Costa (Food Bioscience, 62, 105106, 2024) and Cayman (5-bromo-5-nitro-1,3-Dioxane (CAS 30007-47-7) 2025, as previously relied upon)
Regarding claims 1, and 11-13, Saenton teaches preparing dead bacteria (i.e. treated analytical sample) by suspending (i.e. incubating) viable cells in a solution containing bromo-nitro-dioxane (i.e. antimicrobial). Saenton also teaches diluting cells in ammonium chloride (i.e. control analytical sample). See the paragraph spanning pages 1765-1766. Saenton teaches injecting S. epidermidis bacteria into a sedimentation FFF (SdFFF) channel. Saenton teaches obtaining the fractogram elution profile of the S. epidermidis elution. See p. 1769 para. 1. The sedimentation FFF device includes a UV-Vis detector, which is used to monitor the eluting materials. See p. 1766 para. 1. Furthermore, Saenton discloses that for SdFFF a centrifugal field (i.e. multigravitational field) forces the sample to the accumulation wall. See p. 1765 para. 2. Saenton, in figure 2(a), teaches the fractogram (i.e. elution profile) of dead S. epidermidis and dead P. putida obtained from SdFFF where each peak corresponds to a retention time. Saenton indicates that the SdFFF uses a stopflow time of 2 minutes. See figure 2 (a) caption. Saenton discloses that stopflow relaxation is a procedure whereby a sample is allowed to form equilibrium layers before initiating the elution processes. See p. 1769 para. 2. Furthermore, Saenton teaches comparing the elution profiles and retention times of live bacteria to their dead counterparts. See page 1771 first paragraph. The retention ratio R can be obtained from the fractogram by using the relationship R=t0/tr where tr is the retention time and t0 is void time (i.e. dead time). Saenton discloses that live cells always elute first. See page 1771 first full paragraph. Thus, Saenton teaches determining that the microorganism is susceptible to at least one antimicrobial if their retention time is greater than the retention time of the live cells.
Saenton does not teach at least one microbial population from a biological sample.
Janča teaches using micro-thermal field-flow fractionation for the retention of Staphylococcus epidermidis bacterial cells. See the abstract. The S. epidermidis is isolated from human skin (i.e. biological sample). See section 3.4 para. 2.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Janča S. epidermidis microbial population from a biological sample for Saenton’s S. epidermidis. One would have been motivated to do so because Janča suggests performing field-flow fractionation on the S. epidermidis isolated from human skin. There would have been a reasonable expectation of success because Saenton demonstrates performing sedimentation field-flow fractionation on S. epidermidis.
Saenton and Janča do not teach calculating a percentage of variation in the retention factor between the treated analytical sample and the control analytical sample(s) and comparing the calculated percentage of variation to a significance threshold, the significance threshold being defined for the microorganism and the at least one antimicrobial as a function of an active dose of the at least one antimicrobial, and determining that the microorganism of the microbial population is susceptible to the at least one antimicrobial when the calculated percentage of variation is greater than the significance threshold.
Bégaud-Grimaud teaches a HEL (human erythroleukemia) cell line that is either exposed to diosgenin (i.e. antimicrobial) to form diosgenin treated cells or not exposed to diosgenin to form control cells. After the cells are incubated [with diosgenin], the cells are washed and suspended in sterile PBS prior to sedimentation field-flow fractionation (SdFFF) elution. See p. 9126 section 2.1 para. 1. The SdFFF device includes a separation channel. See section 2.2 para. 1. Bégaud-Grimaud discloses that the HEL cells are eluted from the SdFFF device to which a multi-gravitational field is applied. The elution comprises a mobile phase through the separation channel. See section 2.3. Bégaud-Grimaud teaches UV detection. See table 1 caption. Bégaud-Grimaud indicates that the UV detection is part of the SdFFF device. See fig. 1. Bégaud-Grimaud teaches obtaining fractograms that show a profile with a separation peak corresponding to the cell elution peak. See p. 9128 left column. The different average velocities, and retention order of different species are compared by means of the observed retention ratio Robs where retention ratio Robs =void time (i.e. dead time) versus retention time=t0/tR. See section 3.3 para. 1. Peak shift is quantified by the mean of Robs variation. A reproducible CV<2% (i.e. variation percentage) decrease in Robs after 24 and 48 h incubation is obtained. See p. 9131 right column first passage. In table 3, Bégaud-Grimaud teaches comparing CV percentages between control and treated cells. Bégaud-Grimaud teaches that diosgenin according to the concentration (i.e. active dose) is able to induce apoptosis or differentiation. See section 3.4 para. 1. As such, Bégaud-Grimaud teaches determining whether a cell population experiences apoptosis (death) or differentiation in response to the diosgenin antimicrobial and comparing calculated percentages of variations in table 3. Diosgenin has antibacterial activities, as evidenced by Costa (See p. 2 para. 2).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply Bégaud-Grimaud’s percentage of variation calculation to the elution profiles of Saenton and Janča. One of ordinary skill in the art would have been motivated to apply Bégaud-Grimaud’s suggests calculating the CV of the Robs to determine the reproducibility of the biological effect (see p. 9131 right column first passage). There would have been a reasonable expectation of success because Saenton teaches the same Robs calculation as Bégaud-Grimaud, and Bégaud-Grimaud further teaches obtaining the CV from the Robs values.
Regarding claim 2, 4, 7, 9, 18-19 Janča teaches isolating Staphylococcus epidermidis bacterial cells from human skin and enriching the strain (i.e. reference strain). See section 3.4 para. 2.
Regarding claim 3 and 5, Saenton teaches suspending cells in bromo-nitro-dioxane. See the paragraph spanning pages 1765 to 1766. Bromo-nitro-dioxane is effective against bacteria, as evidenced by Cayman (see the product description).
Bégaud-Grimaud teaches exposing to diosgenin to form diosgenin treated cells. See p. 9126 section 2.1 para. 1. Diosgenin has antibacterial activities, as evidenced by Costa (see p. 2 para. 2).
Regarding claim 10, Bégaud-Grimaud teaches a 3 hour incubation of cells with either a stock solution of diosgenin or an equivalent amount of a vehicle control. See section 2.1 section 1.
Saenton, Bégaud-Grimaud, and Janča do not teach the incubation step with the at least one antimicrobial last from 30 to 120 minutes.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize the time of Saenton’s antimicrobial incubation step based on Bégaud-Grimaud’s suggestion. One of ordinary skill in the art would have been motivated to do so because Bégaud-Grimaud suggests a 3-hour incubation step. There would have been a reasonable expectation of success because Saenton demonstrates incubating cells with the bromo-nitro-dioxane antimicrobial prior to SdFFF, and Bégaud-Grimaud teaches a 3 hour incubation time from which one could reasonably optimize.
Regarding claim 14, Saenton teaches comparing the elution profiles and retention times (i.e. tr ) of live bacteria injected into a FFF channel to their dead counterparts. See page 1771 the first paragraph.
Regarding claim 20, Janča teaches isolating Staphylococcus epidermidis bacterial cells. See section 3.4 para. 2.
Regarding claim 24, Bégaud-Grimaud teaches external field strength from 30 to 200 g, which overlaps with the instantly claimed 10 to 50 g. See p. 9128 left column. Bégaud-Grimaud suggests that if flow rate is sufficiently decreased to offset lift forces, cells are confined to a thin layer and in contact with the accumulation wall. See section 3.3 para. 1.
Saenton teaches a stopflow time of 2 minutes. See figure 2 (a) caption.
Regarding claim 25, Janča teaches isolating Staphylococcus epidermidis bacterial cells. See section 3.4 para. 2.
Saenton discloses that S. epidermidis has an average diameter of 0.9 µm, which is less than 2 µm as instantly claimed. See p. 1769 para. 4.
Claims 8, 16-17 and 22-23 are rejected under 35 U.S.C. 103 as being unpatentable over Saenton, (Separation Science and Technology, 2000, 35(11), 1761-1775), Janča (Journal of Chromatography B, 852(1-2), 512-518, 2007), and Bégaud-Grimaud (Journal of Chromatography A, 1216(52), 9125-9133, 2009), applied to claims 1-5, 7, 9-14, 18-20 and 24-25 above, and further in view of Vaz (US 2014/0199703).
Regarding claim 8, Janča teaches isolating Staphylococcus epidermidis bacterial cells from human skin and enriching the strain (i.e. reference strain). See section 3.4 para. 2.
Saenton, Janča and Bégaud-Grimaud do not teach a biological sample that is urine, stool, sputum, broncho-alveolar washing, pus, cerebrospinal fluid or blood.
Vaz teaches biological samples including urine (i.e. human or animal origin), a blood culture. See claim 6 of Vaz. Vaz teaches a method for detecting in a biological sample comprising microorganisms the presence of microorganisms resistant to a predetermined therapeutic agent comprising: inoculating the sample, on a first tube with, and on a second tube without, at least one such therapeutic agent and incubating it; and performing flow cytometric analysis to obtain one or more flow cytometric parameters for each of the two tubes. See claim 2 of Vaz. Vaz teaches microorganisms including Staphylococcus. See [0037].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Vaz’s biological sample with Janča Staphylococcus biological sample. One of ordinary skill in the art would have been motivated to do so because Vaz suggests that the samples are suitable for flow cytometric analyses. There would have been a reasonable expectation of success because the Staphylococcus epidermidis bacterial cells from human skin taught by Janča and the Staphylococcus from urine, or a blood culture reasonably serve the same function.
Regarding claim 16, Saenton teaches a thin, ribbonlike, open channel in which the separation is induced and controlled by an external field acting in a direction perpendicular to laminar flow in the channel. See page 1763 the third paragraph. Furthermore, Saenton teaches an ethanol aqueous solution bromo-nitro-dioxane (e.g. antibiotic). See the paragraph spanning pages 1765 to 1766.
Saenton, Janča and Bégaud-Grimaud do not teach a kit.
Vaz teaches the concept of a kit for carrying out a method. See claim 23 of Vaz. Vaz teaches a kit for detecting the resistant microorganisms to a therapeutic agent. See [0041].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to organize the bromo-nitro-dioxane (e.g. antibiotic) and the channel of Saenton into a kit, as suggested by Vaz. One would be motivated to organize the components into a kit because Vaz suggests that kits may be useful for carrying out a method. There would be a reasonable expectation of success because Saenton teaches components of a sedimentation FFF system, and Vaz teaches the concept of a kit.
Regarding claim 17, Saenton teaches a sedimentation FFF (SdFFF) system that includes a channel. See page 1766 first full paragraph. For SdFFF, Saenton discloses that the channel is inserted into a centrifuge basket and spun to generate the centrifugal field (i.e. external field). See page 1765 the fifth paragraph. Furthermore, Saenton teaches an ethanol aqueous solution bromo-nitro-dioxane (e.g. antibiotic). See the paragraph spanning pages 1765 to 1766.
Saenton, Janča and Bégaud-Grimaud do not teach a kit.
Vaz teaches the concept of a kit for carrying out a method. See claim 23 of Vaz. Vaz teaches a kit for detecting the resistant microorganisms to a therapeutic agent. See [0041].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to organize the bromo-nitro-dioxane (e.g. antibiotic) and the SdFFF channel of Saenton into a kit, as suggested by Vaz. One would be motivated to organize the components into a kit because Vaz suggests that kits may be useful for carrying out a method. There would be a reasonable expectation of success because Saenton teaches components of a sedimentation FFF system, and Vaz teaches the concept of a kit.
Regarding claim 22, Saenton teaches a FFF channel that uses a semipermeable membrane. See page 1773 the first full paragraph. Saenton teaches a carrier solution with deionized water containing surfactant and bactericide sodium azide (i.e. at least one solution for cleaning). Saenton teaches flushing the channel after each sample injection to clean it. See page 1766 the first full paragraph.
Regarding claim 23, Saenton teaches a carrier solution with deionized water containing surfactant and bactericide sodium azide (i.e. at least one solution for cleaning). Saenton teaches flushing the channel after each sample injection to clean it. See page 1766 the first full paragraph.
Response to Arguments
Applicant's arguments filed 07/10/2026 have been fully considered to the extent that they apply to the new grounds of rejection set forth above but they are not persuasive.
Rejection under 35 U.S.C. 103
Applicant argues that neither Saenton nor Vaz teaches or suggests a stop-flow focusing step in which the mobile phase flow is interrupted so as to concentrate the species against the accumulation wall under a multigravitational field. See p. 23 para. 1.
This argument is not persuasive because Saenton teaches a 2 minute stopflow process for SdFFF. See the caption of figure 4. Bégaud-Grimaud indicates that such flow rate decrease can confine cells (i.e. concentrate) to a thin layer and in contact with the accumulation wall [of the separation channel of the SdFFF device to which multigravitational field is applied] (see section 3.3 para. 1).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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