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 19-20 are new. Claims 1-20 are pending. Claims 1-10, and 15-18 are withdrawn. Claims 11-14 and 19-20 are under consideration in this action.
Priority
Applicant' s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994)
The disclosure of the prior-filed application, Application No. 62/880,375, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Claim 11 recites “steps (a) - (f) are completed within 2 hours of isolation of the bacteria from the patient sample”, which is not supported in 62/880,375, as originally filed and which is required by claims 12-14 and 19-20.
Accordingly, claims 11-14 and 19-20 will receive the effective filing date of 07/30/2020, which is the filing date of PCT/US20/44301.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
New Matter
Claims 11-14 and 19-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. The amendment filed on 12/03/2025 has introduced new matter into the claims.
Claim 11, as filed on 12/03/2025, recites a method of testing antibiotic susceptibility of bacteria isolated from a patient sample, the method comprising: (a) immobilizing bacteria isolated from a patient sample onto a receiving surface; (b) measuring the FLIM signatures at an initial time point upon or prior to contacting the immobilized bacteria with a test antibiotic and at a plurality of intervals, wherein the intervals last for 30 minutes to 1 hour; (c) directing a series of nanosecond excitation pulses of light at the immobilized bacteria; (d) collecting time-correlated fluorescence emitted from individual bacteria immobilized on the receiving surface; (e) generating FLIM-phasor profiles by taking the sine and cosine transform of the fluorescence intensity decays, thereby generating s and g values; and(f) comparing FLIM-phasor profiles obtained before and after the initial time point of step (b); wherein a change in the g value upon contact with a test antibiotic indicates bacterial susceptibility to the test antibiotic, wherein steps (a) - (f) are completed within 2 hours of isolation of the bacteria from the patient sample.
Claim 20, as filed 12/03/2025, recites the method of claim 11, wherein steps (a) - (f) are completed within 1 hour.
Applicant’s amendment, filed 12/03/2025, directs support to paragraphs [0191], [0194], and [0201] and example 4, and asserts that no new matter has been added. See paragraphs 2 and 6 on page 5 of the remarks filed 12/03/2025. However, the specification does not provide sufficient written description of the above underlined limitations.
Claim 11 and dependent claims thereof contain new matter because of the limitation requiring steps (a) - (f) to be completed within 2 hours of isolation of the bacteria from the patient sample. Claim 20 contains new matter because the instant disclosure does not clearly provide support for completing the steps (a)-(f) recited in claim 11 within 1 hour.
The specification as filed and the original claims do not provide support for the limitation in claim 11. The specification teaches the development of a FLIM-AST device for the rapid determination of the antibiotic susceptibility of bacteria. This device can determine antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples. See [0191]. However, steps (a)-(f), as instantly claimed, do not require the use of a particular FLIM-AST device; and the specification suggests that other technology may require between 4 to 12 hours (see [0193] and [0196]). The specification teaches that the invention decreases the time required for diagnosis, with a target of assessment within 2 hours. See [0194]. Yet, paragraph [0194] suggests that such time reduction is attributed to the FLIM-AST device. Furthermore, paragraph [0201] teaches a process that involves: isolating bacteria from patient samples, immobilizing bacteria on agarose pads that containing antibiotics, and imaging using the FLIM-AST device immediately and at 15-minute intervals for 2 hours to assess metabolic kinetics. However, the process described in paragraph [0201] excludes steps recited in the instant claims. In example 4, the specification teaches experiments that determine a distinction between antibiotic resistant and susceptible bacteria for a range of antibiotics after 1 hour. See [0225]. However, example 4 is narrower compared to the instantly claimed method, and example 4 excludes an immobilization step. As such, the specification does not provide support for the newly added limitations in claim 11, because the specification does not teach performing instantly claimed steps (a)-(f) within 2 hours after bacteria have been isolated from patient samples.
The specification as filed and the original claims do not provide support for the limitation in claim 20. The specification states “[i]n some embodiments, the method is completed in less than one hour”. See [0010]. However, the specific method embodiment being referenced in paragraph [0010] is unclear. Paragraph [0009] describes “a method of testing antibiotic susceptibility of bacteria isolated from a patient sample. In some embodiments, the method comprises: (a) immobilizing bacteria isolated from a patient sample onto a receiving surface; (b) contacting the immobilized bacteria with a test antibiotic and measuring the FLIM signatures at an initial time point and at regular intervals for 30 minutes to 1 hour. No addition of fluorophore is required. In some embodiments, the method further comprises: (c) directing a series of nanosecond excitation pulses of light at the immobilized bacteria; (d) collecting time- correlated fluorescence emitted from individual bacteria immobilized on the receiving surface; (e) generating FLIM-phasor profiles by taking the sine and cosine transform of the fluorescence intensity decays, thereby generating s and g values; and (f) comparing FLIM- phasor profiles obtained before and after the contacting of step (b)”. Thus, the specification does not clearly support completing the claimed steps (a)-(f) in one hour.
Such limitations recited in the instant claims 11 (and dependent claims) and 20, which did not appear in the specification or original claims, as filed, introduce new concepts and violate the description requirement of the first paragraph of 35 U.S.C 112. Applicant is required to provide sufficient written support for the limitations recited in the instant claims. Applicant can remove the new matter limitations from the claims to obviate this rejection.
Scope of Enablement
Claims 11-14 and 19-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a FLIM device (as described in paragraph [0203] of the instant specification and below) that can determine antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples; the specification does not reasonably provide enablement for completing steps (a)-(f) within 2 hours of isolation of the bacteria from the patient sample. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention:
The claims are drawn to a method of testing antibiotic susceptibility of bacteria isolated from a patient sample, wherein the method comprises: (a) immobilizing bacteria isolated from a patient sample onto a receiving surface; (b) measuring FLIM signatures and contacting the immobilized bacteria with a test antibiotic; (c) directing a series of nanosecond excitation pulses of light at the immobilized bacteria; (d) collecting time-correlated fluorescence emitted from individual immobilized bacteria; (e) generating FLIM-phasor profiles; and (f) comparing the FLIM-phasor profiles, wherein steps (a)-(f) are completed within 2 hours of isolation of the bacteria from the patient sample. Since the patient sample, bacteria, and antibiotic are not limited in anyway, the claims encompass a variety of biological samples, bacteria with varying replication times, and antibiotics including bactericidal and bacteriostatic antibiotics. Furthermore, the method does not require the use of any particular FLIM device.
According to the instant specification, the current technology to distinguish between resistant and non-resistant strains requires between 4 to 12 hours and a relatively large volumes of blood (10-20 mL) or patient specimen. See [0193]. Most devices on the market rely on the measurements of growth, which requires 4-16 hours due to the requirement for bacteria to replicate. See [0196].
The amount of direction provided by the inventor and the existence of working examples:
Example 1 of the specification teaches monitoring fluorescence lifetimes of NAD(H) production in Pseudomonas aeruginosa and Dictyostelium discoideum populations from the same culture during the growth transition between 4-6 hours. See [0084]. However, example 1 is not an antibiotic susceptibly test.
In example 2, the specification teaches culturing P. aeruginosa strains overnight in PS:DB, diluting, and culturing for 4 to 6 hours. Alternatively, the strains are cultured overnight in a minimal medium, diluted and cultured to an optical density OD600 of 0.2. See [0167]. For experiments involving FLIM measurements of the effects of antimycin A, P. aeruginosa is grown in minimal medium supplemented with antimycin A, cultured to an OD600 of 0.2 [time not disclosed], and immediately measured for fluorescence lifetimes. Fluorescence lifetimes for pyocyanin and pyoverdine are measured. See [0169]. For experiments in which surface-attached strains are cultured in PS:DB media treated with antimycin A, the antimycin A is added after 3 hours of growing following a dilution from an overnight culture. See [0176]. Although the specification suggests that P. aeruginosa is responsible for lung infections in cystic fibrosis patients (see [0043]), the specification does not disclose whether the P. aeruginosa (i.e. the PA14 strain referred to as aphz1/2) used in example 2 was isolated from patient samples.
Example 3 teaches developing a FLIM-AST device for the rapid determination of the antibiotic susceptibility of bacteria. This device can determine antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples. See [0191]. The specification teaches developing a device based on the principles of epi-fluorescence and time-correlated FLIM containing a collection apparatus that correlates the excitation pulse and data collection with nanosecond resolution. See [0200]. The antibiotic susceptibility determination is performed by comparing FLIM-phasor profiles during the 2-hour incubation using software. Cells that are susceptible to antibiotics will exhibit an increase in a shift towards higher g values, as observed in the preliminary data, whereas resistant bacteria will not exhibit such shift. See [0202]. The FLIM microscope uses an 80 MHz ultrafast Ti:Sapphire Mai Tai laser (Spectra- physics, Santa Clara, CA) set at 740 nm for excitation. The setup used a 690-nm SP dichroic- 460/80 nm filter pair for separating emission and a PlanApo N Olympus oil immersion 60X (1.42 NA) objective (Olympus, Waltham, Massachusetts), which is capable of bacterial single-cell resolution. An H7422P-40 photomultiplier tube module (Hamamatsu, Bridgewater, NJ) and A320 FastFLIM Box (ISS, Champaign, IL) were used to measure fluorescence lifetime. Image acquisition was controlled by SimFCS software (Laboratory for Fluorescence Dynamics, Irvine, CA). See [0203]. To measure the fluorescence lifetimes of planktonic and surface-attached P. aeruginosa populations, individual colonies are inoculated into culture tubes containing PS:DB, cultured overnight, diluted, cultured between 1 to 8 hours, and harvested. Planktonic cells are isolated from cultures and immobilized by placing a pad on top of the cells. Similarly, a pad is placed on top of isolated surface-attached cells. Cells are then immediately imaged using the FLIM device. See [0207]. Thus, example 2 does not teach isolating bacteria from any particular patient sample, nor does example 2 exemplify a 2-hour antibiotic susceptibility determination.
In example 4, the specification performing antibiotic susceptibility assessments using 3 clinical E. coli strains from septic patients and 10 clinical S. aureus clinical strains. See [0227]. The bacterial strains are treated using a range of antibiotic concentrations for 1 hour. See [0222]. The g values are computed using the FLIM phasor method, and tetracycline exhibited the greatest changes in g values after 1 hour of treatment. See [0223]. The specification teaches that distinction between antibiotic resistant and susceptible bacteria can be assessed for a range of antibiotics after 1 hour with antibiotic. See [0225] and [0226]. However, the specification is silent regarding the time between isolating the clinical strains and the antibiotic susceptibility determination; furthermore, it is unclear whether the strains are immobilized in example 4.
The state of the prior art and the level of predictability in the art:
With respect to the state of the art of antibiotic susceptibility testing using FLIM, Rojas-Andrade (ACS Infectious Diseases, 2024, 10(12), 4057-4065), a past filing date reference, teaches: isolating E. coli from urinary tract patients, culturing the E. coli overnight (~16 h), immobilizing the cell suspension using LB agar pad, exposing the bacteria to antibiotics, and imaging the bacteria for FLIM analysis. See the first paragraph of the methods section. Rojas-Andrade suggests that bactericidal antibiotics differ in their effect on bacterial metabolic pathways. A longer exposure time, >2h is required to observe significant differences in g-values. See the first paragraph on page 4062. Rojas-Andrade discloses that current phenotypic assays relying on cell growth are the clinical standard for AST, but they require long time periods for microorganisms to demonstrate differentiable growth. See the first paragraph of the introduction. Rojas-Andrade teaches that by tracking shifts in FLIM signal as a function of time and antibiotic concentration, resistant and susceptible phenotypes can be identified in under 1 hour. See the first full paragraph on page 4058. However, this 1-hour time frame taught by Rojas-Andrade does not include the culturing time after isolating the bacterium from the patient sample, or the antibiotic exposure time. Bhattacharjee (as previously relied upon) teaches growing Pseudomonas aeruginosa wild type clinical isolate and Staphylococcus epidermidis ATCC 14990 for 5 hours. See paragraph 4 on page 7 and table S1. Bhattacharjee teaches casting the bacterial solution on a glass coverslip (receiving surface) by spin coating for immobilization, incubating the coverslip with antibiotic and performing FLIM. See paragraph 4-6 on page 7 and paragraph 3 on page 8. As such, Bhattacharjee and Rojas-Andrade teach culturing bacteria isolated from patient samples prior to being immobilization for FLIM measurements.
The quantity of experimentation needed to make or use the invention:
In view of the nature of the invention, the breadth of the claims, the guidance and
working examples in the specification, and the level of predictability within the art, as
evidenced above, one of skill in the art could not complete the claimed antibiotic susceptibility test within 2-hours of isolating a bacterium from a patient sample, without undue experimentation. To date, the clinical standard for AST relies on cell growth, as evidenced by Rojas-Andrade. The instant specification acknowledges that most AST devices require 4-16 hours due to the requirement for bacteria to replicate. The instant specification does not provide a working example in which steps, commensurate in scope with claimed (a)-(f), are completed within 2 hours after bacteria have been isolated from patient samples. Consequently, there is no indication that the claimed method can test the antibiotic susceptibility of any bacterium from any patient sample within 2 hours of isolation, which is commensurate with the scope of the claims.
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 11-14 and 19-20 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 11 recites “(b) measuring the FLIM signatures at an initial time point upon or prior to contacting the immobilizing bacteria with a test antibiotic and at a plurality of intervals, wherein the intervals last for 30 minutes to 1 hour”, which is indefinite because it is unclear when the FLIM signatures are required to be measured relative to the antibiotic contact step. To clarify, step (b) as amended requires measuring the FLIM signatures either upon contacting the immobilized bacteria with a test antibiotic, or prior to such contact; and measured at a plurality of intervals wherein the intervals last for 30 minutes to 1 hour. If the FLIM signature is measured prior to the contacting step, it is unclear whether the intervals are also required to be prior to the contact step. Furthermore, if the FLIM signature is measured prior to the contact step, then it is further unclear which “initial time point” is being referenced in step (f).
Claims 12-14 depend from claim 11 and are rejected for the reason set forth above.
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 13-14 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.
Claims 13 and 14 do not clearly limit claim 11 from which they depend. Claim 11 requires steps (a)-(f) to be completed within 2 hours of isolation of the bacteria from the patient sample. Claim 13 broadens the scope of this 2-hour time frame because claim 13 requires steps (c)-(e) to be repeated at intervals of 10-20 minutes for 1-3 hours after contacting the bacteria with test antibiotic. Moreover, claim 14 does not clearly include all the limitations of claim 11, because claim 14 requires steps (c)-(e) to be repeated at intervals of 15 minutes for 2 hours after contacting the bacteria with test antibiotic.
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 Interpretation
Claim 11 requires six method steps. First, bacteria, isolated from a patient sample, are immobilized onto any receiving surface. Second, an FLIM signature measurement is made simultaneously with or before contacting the immobilized bacteria with a test antibiotic, and the FLIM signature is also measured multiple times thereafter for an interval of 30 minutes to 1 hour. Third, a series of nanosecond excitation pulses of light are directed at the immobilized bacteria. For example, specification teaches an FLIM microscope that uses 80 MHz ultrafast Ti:sapphire Mai Tai laser set at 740 nm for excitation. See [0203]. Fourth, time-correlated fluorescence emitted from individual bacteria immobilized on the receiving surface is collected. For example, the instant specification teaches a H7422P-40 photomultiplier tube module (Hamamatsu, Bridgewater, NJ) and A320 FastFLIM Box (ISS, Champaign, IL) to measure fluorescence lifetime; and controlling image acquisition with SimFCS software (Laboratory for Fluorescence Dynamics, Irvine, CA). See [203]. Fifth, FLIM-phasor profiles are generated by taking the sine and cosine transform of the fluorescence intensity decays, thereby generating s and g values. The equation for generating s and g values requires taking the sine and cosine transform of the fluorescence intensity decays. See paragraph [0055] of the instant specification for the equations used for deriving the s and g values. Sixth, FLIM-phasor profiles, e.g. s and g values, are compared before and after the immobilized bacteria are contacted with the test antibiotic. As discussed above, the claims are enabled for using the FLIM device described in paragraph [0203] of the instant specification, which is disclosed as being capable of determining antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples.
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 11, 13-14 and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Bhattacharjee (Scientific reports, 2017, 7(1), 3743), with evidence from Banner (Journal of bacteriology, 2007 189(7), 2793-2804) and Ranjit, (Nature protocols, 2018 13(9), 1979-2004). Bhattacharjee is cited on the IDS filed 04/28/2022, however it is included with the supplementary information herein.
Regarding claim 11, Bhattacharjee teaches using an NAD(P)H FLIM-phasor technique to differentiate metabolic states of live bacterial populations at the single-cell level. See paragraph two on page 2. Bhattacharjee teaches growing bacterial strains, including E. coli, Pseudomonas aeruginosa wild type clinical isolate and Staphylococcus epidermidis ATCC 14990, in lysogeny broth (LB). See paragraph 4 on page 7 and table S1. Evidentiary reference Banner discloses that S. epidermidis ATCC 14990 is a nasal isolate. See the third paragraph in the left column on page 2794 of Banner. For imaging, Bhattacharjee teaches preparing samples by mixing LB shaking cultures with 1% agarose and casting the resulting solution on a glass coverslip (receiving surface) by spin coating. See paragraph 4 on page 7. The agarose-embedded bacterial samples on glass coverslip (immobilized bacteria) are incubated in nalidixic acid or ampicillin (test antibiotics) for 30 min. The samples are washed, incubated in LB media and imaged. See paragraphs 5-6 on page 7. For growth curve sample preparation, LB media is inoculated with bacteria, one culture aliquot is taken for each bacterium every 2 hours and samples for FLIM measurements are prepared. See the last paragraph on page 7. Fluorescence lifetime imaging microscopy (FLIM) is performed on a Zeiss LSM 710 microscope coupled to an 80 MHz multiphoton excitation laser source (i.e. a series of 0.08 cycles per nanosecond laser light pulses). A photomultiplier tube H7422P-40, Hamamatus, Japan is used as the microscope external detector port photo-sensor unit (collector of florescence correlated with time, specifically lifetime) and a A320 FastFLIM tube is employed to acquire frequency domain FLIM data. SimFCS software is used for FLIM data acquisition. The g, s coordinate system is used to indicate phasor cursor coordinates (FLIM-phasor profiles) that use the first harmonic phasor plots at 80 MHz. See paragraph 3 on page 8 and figure S1 for the images of single bacteria. The phasor coordinates (FLIM-phasor profile) of each bacterial cell are represented by a single datapoint and show variations along both g and s axes of the phasor plot. See paragraph two on page 2 and figure 1a. Evidentiary reference Ranjit discloses that phasor coordinates are calculated using the following relations: gi,j (ω)= T∫0 I(t)∙cos(nωt)dt / T∫0 I(t)dt and si,j (ω)= T∫0 I(t)∙sin(nωt)dt / T∫0 I(t)dt. See the bottom of page 1980 of Ranjit. Bhattacharjee discloses that E. coli cells treated with nalidixic acid shift towards longer fluorescence lifetimes with increasing concentration, as compared to untreated control. E. coli cells similarly exposed to increasing concentrations of ampicillin exhibit corresponding phasor position shifts to larger g values, while s values remain relatively constant. See the last full paragraph on page 2. Thus, Bhattacharjee suggests that this change in the g value upon contact with a test antibiotic indicates bacterial susceptibility to antibiotic, as shifts of the phasor position of cells treated with antibiotics corresponds to a concentration-dependent increase in the fraction of dead cells. See the last full paragraph on page 2. Furthermore, the FLIM system taught by Bhattacharjee (paragraph 3 page 8) is indistinguishable from that which is instantly disclosed; therefore the FLIM system of Bhattacharjee is considered to be inherently capable of determining antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples. See MPEP 2112.01(II).
Bhattacharjee does not teach (b) measuring the FLIM signatures at an initial time point upon contacting the immobilized bacteria with a test antibiotic and at a plurality of intervals for 30 minutes to 1 hour; (f) FLIM-phasor profiles obtained before and after initial time point of step (b).
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 rearrange the order of the steps taught by Bhattacharjee and to further optimize the timing of the FLIM measurements by adjusting the 2-hour time interval. In the process, one could reasonably arrive at a process in which the FLIM measurement step is performed before the nalidixic acid or ampicillin incubation step, and performed multiple times thereafter at an interval. One would be motivated to perform an FLIM measurement before the incubation step because Bhattacharjee suggests that phasor of recovered cells [recovered from antibiotic exposure] shift to significantly longer lifetimes than the initial population before exposure to nalidixic acid. See the figure 3b and the first full paragraph on page 4. Thus, one would be motivated to take an initial FLIM measurement for a lifetime comparison. There would be a reasonable expectation of success because Bhattacharjee demonstrates comparing bacterial phasor response to antibiotic treatment (e.g. see figure 2). One would be further motivated to decrease the 2-hour of Bhattacharjee because Bhattacharjee suggests that the NAD(P)H lifetime phasor distribution changes non-monotonically as a function of growth phase. There would be a reasonable expectation of success because Bhattacharjee demonstrates acquiring FLIM data from agarose-embedded samples at multiple intervals. MPEP 2144.05(II) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claims 13-14, Bhattacharjee teaches the agarose-embedded bacterial samples on glass coverslip (immobilized bacteria) are incubated in nalidixic acid or ampicillin (test antibiotics) for 30 min. The samples are washed, incubated in LB media and imaged. See paragraphs 5-6 on page 7. For growth curve sample preparation, LB media is inoculated with bacteria, one culture aliquot is taken for each bacterium every 2 hours and samples for FLIM measurements are prepared. See the last paragraph on page 7. Thus, Bhattacharjee indicates that the method is repeated for each bacterium every 2 hours in order to prepare the growth curve.
Bhattacharjee does not teach repeating instant steps (c)-(e) at intervals of 10-20 minutes for 1-3 hours after contacting the bacteria with the test antibiotic (relevant to instant claim 13).
Bhattacharjee does not teach repeating instant steps (c)-(e) at intervals of 15 minutes for 2 hours after contacting the bacteria with the test antibiotic (relevant to instant claim 14).
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 timing of the FLIM measurements by adjusting the 2-hour time interval. One would be motivated to optimize the 2-hour interval of Bhattacharjee because Bhattacharjee suggests that the NAD(P)H lifetime phasor distribution changes non-monotonically as a function of growth phase. See paragraph 2 on page 2. There would be a reasonable expectation of success because Bhattacharjee demonstrates acquiring FLIM data at multiple intervals. MPEP 2144.05(II) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 19, Bhattacharjee teaches incubating agarose-embedded bacterial samples on glass coverslip in nalidixic acid or ampicillin (i.e. a penicillin-type antibiotic) for 30 min. See paragraphs 5-6 on page 7.
Regarding claim 20, Bhattacharjee teaches casting culture solutions on a glass coverslip by spin coating for 10 seconds. The agarose-embedded samples are incubated with the antibiotic for 30 min and imaged. See the first two paragraphs of the materials and methods section. The image scan speed is 25 µs/pixel. To create bacterial phasors, the average g and s value of the phasor distribution from individual bacterial cell are calculated and plotted. See paragraphs 3 and 5 on page 8. Evidentiary reference Ranjit such calculation includes sine and cosine (the bottom of page 1980). Bhattacharjee discloses that the metabolism of bacterial populations varies drastically over time. See the last paragraph on page 6.
Bhattacharjee does not teach completing instantly claimed steps (a)-(f) within 1 hour.
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 duration of the steps taught by Bhattacharjee. One would be motivated to do so because Bhattacharjee suggests that metabolisms vary over time. There would be a reasonable expectation of success because Bhattacharjee demonstrates a 10 second immobilization step, a 30 min antibiotic contact step, and FLIM measurement steps. Furthermore, Bhattacharjee suggests that the NAD(PH) lifetime phasor data distribution captures metabolic activity that may not be inferred by cell density; which in turn suggests that a full 2-hour growth time may not be necessary. See, for example, bottom passage on page 4 for the 2-hour growth time taught by Bhattacharjee.
(Maintained) Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Bhattacharjee (Scientific reports, 2017, 7(1), 3743), as applied to claims 11 and 13-14 above, and further in view of Jeong (Biomedical optics express, 2018, 9(7), 3434-3448) and Kim, (Fisheries and Aquatic Sciences, 2018, 21(1), 18).
The teachings of Bhattacharjee with respect to instant claim 11 are discussed above.
Regarding claim 12, Bhattacharjee teaches growing bacterial strains, including E. coli, Pseudomonas aeruginosa wild type clinical isolate (e.g. bacteria isolated from patient) and Staphylococcus epidermidis ATCC 14990, in lysogeny broth (LB). See paragraph 4 on page 7 and table S1. For imaging, Bhattacharjee teaches preparing samples by mixing LB shaking cultures with 1% agarose and casting the resulting solution on a glass coverslip (e.g. receiving surface) by spin coating. See paragraph 4 on page 7. The agarose-embedded bacterial samples on glass coverslip (e.g. immobilized bacteria) are incubated in nalidixic acid or ampicillin for 30 min. The samples are washed, incubated in LB media and imaged. See paragraphs 5-6 on page 7.
Bhattacharjee does not teach a test antibiotic selected from the group consisting of: amoxicillin, cephalexin, erythromycin, ciprofloxacin, trimethoprim, tetracycline, and gentamicin.
Jeong teaches a method for the selective visualization of minocycline (i.e. a tetracycline antibiotic) within human skin tissue utilizing fluorescence lifetime imaging microscopy (FLIM). See the abstract and the first sentence of the introduction section. Jeong discloses that minocycline is a tetracycline class antibiotic frequently used for the treatment of acne vulgaris. Topical minocycline gel (BPX-01) is a new drug product that aims to drive minocycline to the microenvironments where P. acnes reside. BPX-01’s mechanism is intended to deliver minocycline through the stratum corneum of the skin into pores and hair shafts. See the paragraph spanning pages 2-3.
Kim discloses that cutaneous bacterial pathogens including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Propionibacterium acnes are often involved in acne vulgaris. See the abstract. Kim suggests that generally, the topical therapeutic option for these cutaneous pathogens is antibiotic treatment to destroy the microbes. However, irrational use of such antibiotics can result in antibiotic-resistant bacteria causing treatment failures and fatal outcomes in various infectious diseases. See the first paragraph of the background section.
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 replace the nalidixic acid or ampicillin of Bhattacharjee with the minocycline of Jeong in view of Kim. One would be motivated to use the minocycline of Jeong because Jeong suggests that minocycline is used to treat acne vulgaris, and Kim suggests that Staphylococcus epidermidis, and Pseudomonas aeruginosa (i.e. two bacteria of Bhattacharjee) are often involved in acne vulgaris. There would be a reasonable expectation of success because Jeong demonstrates visualizing minocycline update with FLIM and Bhattacharjee demonstrates determining the antibiotic susceptibility of bacteria with FLIM.
Response to Arguments
Applicant's arguments filed 12/03/2025 have been fully considered but they are not persuasive.
Applicant argues that the cited references do not teach or suggest all of the limitations of claims 11-14. Bhattacharjee teaches growing bacterial strains for 5 hours, and then taking aliquots for testing. None of the references teach or suggest the use of FLIM to test antibiotic susceptibility without requiring multiple hours of incubation. None of the references teach or suggest that AST can be completed within 2 hours of isolating a bacterial sample from a patient. See the first paragraph on page 7 of the remarks.
This argument is not persuasive because the specification describes a FLIM-AST device that can determine antibiotic susceptibility within 2 hours after bacteria have been isolated from patient samples. See [0191]. Bhattacharjee teaches a substantially identical FLIM system structure compared to the instantly disclosed FLIM-AST device described in paragraph [0203]. Therefore, the FLIM system of Bhattacharjee is inherently capable of determining antibiotic susceptibility within the claimed time frame, absent evidence to the contrary. Furthermore, the instant claim 11 is open-ended due to the term “comprising”. Therefore, the claimed method does not exclude additional steps, such as an incubation step.
Applicant argues that Bhattacharjee does not provide a reasonable expectation of success that one can extrapolate FLIM data from one type of antibiotic to other categories of antibiotics, given that different antibiotics operate via different mechanisms that do not necessarily target bacterial metabolism. See the first paragraph on page 7 of the remarks.
This argument is not persuasive because the rationale discussed above does not rely on extrapolating FLIM data from one type of antibiotic to another. To the extent that Applicant is arguing that the data of Bhattacharjee are not applicable to the antibiotic of Jeong, it is not persuasive. The test for obvious is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Applicant argues that the claimed method can be completed with the critical speed necessary for life-or-death treatment decisions using a variety of bacteria including clinical isolates and a variety of antibiotics (see example 4 of the instant specification and Rojas Andrade(2024)). See the second paragraph on page 7 of the remarks.
This argument is not persuasive because the fact that applicant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
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