DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 are pending in this application.
Applicant’s amendment to the claims filed 01/29/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed on 01/29/2026 in response to the non-final rejection mailed on 10/29/2025 are acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Objections
The objections to the claims are withdrawn in view of
the amendment to claim 1 to no longer recite “and” between steps (iv) and (v), and
the amendment to claim 136 to recite “incubating the cells of the microorganism” in line 4 and “for said cells to undergo only 1 to 5 cell divisions” in lines 10-11.
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention is withdrawn in view of
the amendment to claim 1 to recite “wherein said label is capable of arresting cell division” in step (iii), and
the amendment to claim 11 to no long recite “any combination thereof” referring to the use of multiple nucleic acid binding fluorescent compounds.
Claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 are newly rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
The instant rejection is newly stated and is necessitated by claim amendment.
Claim 1 (claims 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 dependent therefrom) is indefinite for the recitation of “said label” in line 6 of step (iii). There is insufficient antecedent basis for this limitation in the claims.
Claims 11, 41, 69 and 134 are indefinite for the recitation of “the nucleic acid binding fluorescent compound”. There is insufficient antecedent basis for these limitations in the claims.
Response to remarks: Beginning on page 16 of Applicant’s response to rejections under 35 USC 112(b); Applicant in summary contends that the amendments to the claims overcome the 112b rejections of record.
Applicant’s remarks are considered and found not convincing, as the amendments to the claims have necessitated new grounds of rejection above.
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.
Claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 are newly rejected under 35 U.S.C. 112(a) 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, at the time the application was filed, had possession of the claimed invention. This is a new matter rejection and is necessitated by amendment.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims.” See also MPEP 714.02. MPEP § 2163.II.A.3.(b) further states, “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112, para. 1, as lacking adequate written description.” According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117.”
Claim 1 (claims 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 dependent therefrom) has been amended to recite “exposing to a composition consisting of a single labelling compound the actively dividing cells of the microorganism”. According to applicant’s instant remarks in paragraph 1 of the section “Amendments to the claims and support” on page 15, “[support for the amendments can be found in] cancelled claims 128, 129 and 133, and in the description, e.g., at paragraphs [00185]-[00187] of the specification as originally filed”. However, there is no apparent descriptive support for the limitation “exposing to a composition consisting of a single labelling compound the actively dividing cells of the microorganism” in the original application as filed. Absent descriptive support, the noted limitation is considered to introduce new matter into the claims. Applicant is invited to show support for the limitation at issue.
Claim Rejections - 35 USC § 103
Claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 are rejected under 35 U.S.C. 103 as being unpatentable over Madhavan (WO2014037883; cited on the IDS submitted 07/10/2023; herein referred to as Madhavan) in view of Jorgensen et al. (Clin Infect Dis, 2009, 49:1749; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as Jorgensen), Martin et al. (Cytometry Part A, 2005, v 67A:45-52; cited in the Form PTO-892 mailed 12/28/2023; herein referred to as Martin), Papich et al. (Veterinary Microbiol, 2014, v 171: 480-486; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as Papich) and Koch et al. (J Microbiol Meth, 1996, 27:49; cited on the Form PTO-892 mailed 10/29/2025; herein referred to as Koch) as evidenced by Invitrogen at al. (Molecular Probes Product Information, 2005, LIVE/DEAD FungaLight Yeast Viability Kit, p 1-4; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as LD), ATCC et al. (Candida Albicans Robin Berkhout, website https://www.atcc.org/products/10231, published 2023, visited on 12/13/2023; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as ATCC), Attune et al. (Attune Acoustic Focusing Cytometer, 2014, User Guide, Life Technologies; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as ATTUNE) and Dabrowa et al. (Sabouraudia, 1968, v 6(1): 51-56; cited on the Form PTO-892 mailed 12/28/2023; herein referred to as Dabrowa).
The instant rejection is maintained from a previous office action and any newly recited portions are necessitated by claim amendment.
Claim 1 (claims 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 dependent therefrom) is drawn to a method of determining and/or quantifying the susceptibility of a unicellular or multicellular microorganism to an antimicrobial agent by acoustic flow cytometry and treating a subject having or suspected of having an infection with the unicellular or multicellular microorganism, said method comprising the following steps:
(i) incubating in a culture medium
(a) cells of the microorganism from a sample obtained from a subject having or suspected of having an infection with said microorganism, and/or
(b) a sample obtained from the subject having or suspected of having an infection with said microorganism, wherein said sample comprises cells of said microorganism,
wherein said incubating is for a time and under conditions sufficient for said cells to actively divide to thereby obtain an actively dividing culture of said cells of said microorganism;
(ii) exposing cells of the microorganism in and/or from the actively dividing cell culture obtained in step (i) to an antimicrobial agent for a time and under conditions sufficient for said cells of the microorganism to actively divide;
(iii) exposing to a composition consisting of a single labelling compound the actively dividing cells of the microorganism that were exposed to the antimicrobial agent in step (ii), resulting in the labelling of the cells with a single labelling compound,
wherein said label is capable of arresting cell division of intact and/or replicating cells of said microorganism,
and wherein said method does not comprise labelling the cells of the microorganism with any other compound;
(iv) measuring effect of said antimicrobial agent on cellular morphology of said cells of the microorganism by acoustic flow cytometry,
wherein said measuring comprises measuring effect of said antimicrobial agent on the cellular morphology of actively dividing cells of said microorganism as said cells become compromised by said antimicrobial agent and before cells lysis or cell breakdown or cell death following exposure of said actively dividing cells of said microorganism to said antimicrobial agent, and
wherein said measuring comprises measuring an increase in size and/or cytoplasmic volume of actively dividing cells of said microorganism following exposure of said cells to a concentration of said antimicrobial agent as determined by acoustic flow cytometry following exposure of said cells to a concentration of said antimicrobial agent, relative to the size and/or cytoplasmic volume of actively dividing cells of said microorganism in and/or from the sample obtained from the subject as determined by acoustic flow cytometry absent exposure of said cells to said antimicrobial agent;
(v) determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, wherein step (v) comprises:
(A) determining susceptibility of the microorganism to the antimicrobial agent qualitatively by determining and/or quantifying from the acoustic flow cytometry data output that said microorganism is either susceptible or not susceptible to said antimicrobial agent; and
(B) determining susceptibility of the microorganism to the antimicrobial agent quantitatively by determining and/or quantifying from the acoustic flow cytometry data output the minimal inhibitory concentration (MIC) of said antimicrobial agent to which the microorganism is susceptible; and
(vi) treating the subject having or suspected of having an infection with the unicellular or multicellular microorganism by administering to the subject a therapeutically effective amount of said antimicrobial agent to which the microorganism is determined and/or quantified to be susceptible in step (v).
Madhavan describes antimicrobial formulations [title] that are suitable for use against bacterial and fungal organisms [p 9, lines 25-27], .
Regarding claim 1 and the limitations of step (i) of incubating in a culture medium cells of the microorganism from a sample obtained from a subject having or suspected of having an infection with said microorganism, Madhavan discloses the process of culture treatment in Example 1 [p 10, lines 16-26] wherein cells are grown in a culture flask of tryptic soy agar per ATCC guidelines for approximately 30 hours to reach 90% confluence. Madhavan similarly applies the method of Example 1 in Example 30 [p 21, lines 8-14] to the fungal organism Candida albicans ATCC 10231 that is a microorganism isolated from a man with bronchomycosis as evidenced by ATCC [p 1, col 1].
Regarding claim 1 and the limitations of step (ii) of exposing cells of the microorganism and/or from the actively dividing cell culture obtained in step (i) to an antimicrobial agent for a time and under conditions sufficient for said cells of the microorganism to actively divide, Madhavan discloses treating the fungal sample with a formulation for 20-180 min along with a corresponding control [p 11, lines 16-18], which is considered to correspond to a time and under conditions sufficient for said cells of the microorganism to actively divide. Additionally, the corresponding control of Madhavan is considered to correspond to a sample incubated in parallel without exposure to the antimicrobial agent.
Regarding claim 1 and the limitations of step (iii) of labelling cells of the microorganism exposed to the antimicrobial agent in step (ii) with a nucleic acid binding fluorescent compound, Madhavan discloses the step of adding contents of the LIVE/DEAD FungaLight Yeast Viability Kit to the fungal cells after exposure to antimicrobial formulation as described in the viability assay protocol of Example 2 [p 11, lines 22-23], wherein the kit contents comprise a mixture of SYTO9 and propidium iodide, both of which are nucleic acid binding fluorescence compounds as evidenced by LD [p 1, col 1, para 1]. As Madhavan discloses “adding 10 ul of the LIVE/DEAD Funga Light Yeast Viability Kit” to the cells and incubated for 5 minutes [Example 2, p 11, lines 22-23], and the kit comprises both SYTO9 and PI with instructions to add compounds both singularly and together to samples as evidenced by LD [p 2, col 2, sections 2.2-2.4], the method of Madhavan is considered to encompass both the addition of both SYTO9 and PI together to samples, as well as SYTO9 and PI individually to separate samples. Therefore, the teachings of Madhavan extend to the labeling of cells by a single nucleic acid binding fluorescent compound, as live cells would only be labeled by SYTO9 considering that PI only interacts with dead cells. Additionally, as the SYTO9 of Madhavan is encompassed by the structural limitations of a nucleic acid binding fluorescent compound set forth in the claim, it is presumed to have the activity of being capable of arresting cell division, as the activity of the compound is presumed to be inherent to its structure (see MPEP 2112.01.I). Additionally, the limitation of “wherein said method does not comprise labeling the cells of the microorganism with any other compound”, the term “the cells” can be broadly interpreted to encompass all of the cells of the microorganism in the sample. In view of this interpretation, as the live cells of Madhavan are only labeled by SYTO9, all of the cells of the microorganism in the sample are not labeled with any other compound.
Regarding claim 1 and the limitations in step (iv) of measuring effect of said antimicrobial agent on cellular morphology of said cells of the microorganism by acoustic flow cytometry, Madhavan discloses the analysis of labeled samples using the Attune flow cytometer [p 10, lines 7-8], which is an acoustic flow cytometer as evidenced by ATTUNE [p 1, title] to mathematically determine the number of cells as a percentage of the control [p 11, lines 24-26].
Regarding claim 1 and the limitations in step (v)(A) of determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, comprising determining susceptibility of the microorganism to the antimicrobial agent qualitatively by determining and/or quantifying from the acoustic flow cytometry data output that said microorganism is either susceptible or not susceptible to said antimicrobial agent, Madhavan discloses the analysis of labeled samples using the Attune flow cytometer [p 10, lines 7-8], which is an acoustic flow cytometer as evidenced by ATTUNE [p 1, title] to mathematically determine the number of cells as a percentage of the control [p 11, lines 24-26]. As SYTO9 is used to label all yeast cells including both cells with intact membranes and those with damaged membranes, and PI is used to label only cells with damaged membranes as evidenced by LD, the comparison of these labeled samples to controls is effectively a measurement of the change in cell morphology as it relates to the damage of cell membranes by the antimicrobial agent. Given the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the method disclosed by Madhavan satisfies this limitation of claim 1.
Madhavan does not teach measuring an increase in size and/or cytoplasmic volume of actively dividing cells in step (iv), the determination of MIC in step (v)(B), and treating a subject having or suspected of having an infection with the unicellular or multicellular microorganism by administering to the subject a therapeutically effective amount of said antimicrobial agent in step (vi).
Jorgensen discusses the principles of antimicrobial susceptibility testing [title], wherein MIC is discussed as the lowest concentration of antibiotic that prevents growth [p 1, col 2, para 1], and that the determination of the MIC provides a quantitative and qualitative assessments of whether an organism is susceptible, intermediate, or resistant to an antimicrobial [abstract].
Regarding claim 1 and the limitations in step (v)(B) of determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, comprising determining susceptibility of the microorganism to the antimicrobial agent quantitatively by determining and/or quantifying from the acoustic flow cytometry data output the minimal inhibitory concentration (MIC) of said antimicrobial agent to which the microorganism is susceptible, Jorgensen teaches the broth dilution test method wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, and after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that prevented growth, which was classified as the MIC [p 1749, col 2, para 1]. As Madhavan teaches the use of acoustic flow cytometry methods to gather data related to the effect of antimicrobial agents on cells through the comparison of nucleic acid amounts of exposed cells to that of unexposed cells, and Jorgensen teaches the method for determining MIC using data related to cell growth as a result of exposure to different concentrations of antimicrobial agent, one of skill in the art would be capable of applying the data produced by the method of Madhavan to determine MIC based on the concepts taught by Jorgensen.
Regarding claim 1 and the limitations in step (vi) of treating a subject having or suspected of having an infection with the unicellular or multicellular microorganism by administering to the subject a therapeutically effective amount of said antimicrobial agent, Jorgensen teaches the results of a susceptibility test such as MIC must be interpreted and reported to a physician, which includes interpretation of the likely success of a particular agent in eradicating bacteria at various body sites [p 1753, col 1, para 4], and that determination of MIC and classification of organisms as resistant, susceptible, or intermediate aid a physician in selecting a particular drug for an effective therapy [p 1753, col 2, para 2]. Therefore Jorgensen teaches the determination of MIC is an important step to aiding the administration of a therapeutically effective treatment of a patient, and therefore implicitly discloses the administration of a therapeutically effective amount of an agent to which a microorganism has been determined to be susceptible.
Considering an alternative interpretation of Jorgensen, wherein Jorgensen does not implicitly disclose the administration of a therapeutically effective amount of an antimicrobial agent, Papich discusses the rational selection of dosage regimes for the prudent use of antimicrobial drugs [title], wherein it is taught that administration must ensure there is sufficient drug to eliminate susceptible isolates [p 480, col 1, para 1].
Regarding claim 1 and the limitations in step (vi), Papich teaches administration must ensure there is sufficient drug to eliminate susceptible isolates [p 480, col 1, para 1], which is achieved by maintaining drug concentrations in serum above the MIC for a portion of the dose interval [p 482, col 1, para 3], which is considered to encompass a therapeutically effective amount of the antimicrobial agent. Papich further provides examples that for treating of a gram-negative infection, especially a serious one, some regimens for penicillins and cephalosporins require administration 3-4 times per day [beginning p 482, col 2, para 5], and that cephalexin and amoxicillin-clavulanate have been used to successfully treat staphylococcal infections when administers once daily.
Considering an alternative interpretation wherein the method of Madhavan is considered not to correspond to the limitations of labeling the cells with a single compound and no other compound, Martin discusses DNA labeling in living cells [title], wherein different fluorescent dyes are discussed in terms of their advantages with different biological applications, such as the use of DRAQ5 for its ability to stain DNA in living cells with low photobleaching and deep red excitation/emission [abstract].
Regarding claim 1 and the limitations in step (iii) of labeling cells with a single nucleic acid dye that results in arrest of cell division and labeling the cells with no other compound, Martin teaches the labeling of cells by staining DNA with DRAQ5 fluorescent dye can hinder cell cycle progression at the G2 phase because it is an anthracycline derivative that intercalates into DNA and conceivably blocks topoisomerase II [p 51, col 1, para 3]. As the method of Madhavan cited above is carried out on the yeast microorganism C. albicans, one of skill in the art would recognize that DRAQ5 would be applicable to arresting cell development in this organism at G2 phase due to its intercalation activity.
Koch relates to the deduction of cell volume and mass for forward scatter intensity of bacteria analyzed by flow cytometry [title], and describes the theory that forward scatter signals from a flow cytometer can be converted to cellular volumes that are applicable to bacteria [abstract], which is an important variable in studies involving kinetic models of growth, nutrient transport and metabolism.
Regarding claim 1 and the limitations in step (vi) of measuring an increase in size and/or cytoplasmic volume of actively dividing cells, Koch teaches a method of using flow cytometry measurements of forward scattering intensity of bacterial samples to calculate cell volume, and teaches that such a method is built on the relationship between forward scatter intensity and cell size [p 50, col 1, para 1]. Koch indicates the distinction of this method is in part due to the size of bacteria being substantially smaller than mammalian cells that were more frequently studied by flow cytometry at the time of the references publication [p 50, col 2, para 1], therefore implying that the assessment of cell volume via flow cytometry was routine for cells larger than bacteria at that time via forward scattering analysis, and that the method presented in the reference made available the application of cell volume measurements on bacteria. Therefore the teachings of Koch involving the use of flow cytometry forward scattering data to assess cell volume are considered to correspond to the limitation of measuring an increase in size and/or cytoplasmic volume of actively dividing cells.
In view of Madhavan, Jorgensen, Papich and Koch, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Madhavan by determining MIC and administering a therapeutically effective amount of an antimicrobial agent, as taught by Jorgensen, administering the antimicrobial agent to a subject with infection, as taught by Papich, and measuring an increase in cell size and/or cytoplasmic volume, as taught by Koch, to arrive at the claimed invention.
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by determining MIC of the tested antimicrobial agent, because Jorgensen teaches that determining MIC provides a qualitative and quantitative assessment of whether an organism is susceptible, intermediate, or resistant to an antimicrobial agent and such determinations can aid a physician in selecting a particular drug for an effective therapy against infection.
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by assessing the cell volumes and sizes via the collected flow cytometry data, because Madhavan teaches a method of assessing growth characteristics of cells treated with antimicrobial agents via flow cytometry, and Koch teaches using flow cytometry data to assess cellular volumes and sizes is an important variable in studies involving kinetic models of growth, nutrient transport and metabolism.
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by administering a therapeutically effective amount of the antimicrobial agent to a subject, because Papich discloses that administration must ensure there is sufficient drug to eliminate susceptible isolates achieved by maintaining drug concentrations in serum above the MIC for a portion of the dose interval, and gives examples of treating gram-negative infection with regimens of penicillins and cephalosporins for 3-4 times per day.
One of ordinary skill in the art would have had a reasonable expectation of success because both Madhavan and Jorgensen discuss techniques for determining the susceptibility of microorganisms to antimicrobial agents, Madhavan and Koch discuss techniques for using flow cytometry data to assess characteristics of cells throughout growth, and Jorgensen and Papich discuss the administration of sufficient amounts of antimicrobials to treat microbial infection as an application of determined antimicrobial susceptibility data.
In view of Martin, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Madhavan, Jorgensen, Papich and Koch by using DRAQ5 as a nucleic acid binding fluorescent compound, as taught by Martin, to arrive at the claimed invention, as the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the SYTO9 and PI of Madhavan and the DRAQ5 of Martin are nucleic acid binding fluorescent compounds, and as such all are capable of being incorporated into such methods as described by Madhavan. Thus it would have been obvious to one of ordinary skill in the art to replace the SYTO9 and PI dyes of Madhavan with the DRAQ5 dye of Martin, as one of ordinary skill in the art would have been able to carry out such a substitution with a reasonable expectation of success because both Madhavan and Martin discuss methods of labeling nucleic acids with fluorescent compounds.
Regarding claim 17, Madhavan discloses the method of determining the percentage of live cells as described in Example 2 [beginning p 10] as a result of the LIVE/DEAD FungaLight Yeast Kit [p 11, lines 22-23] comprising steps of exposure of a fluorescently labeled sample to an antimicrobial agent compared with controls without antimicrobial treatment [p 11, lines 16-18] that contains SYTO9 and PI as evidenced by LD [p 1, col 1, para 1], and Martin teaches the use of DRAQ5 to label nucleic acid [p 51, col 1, para 3]. The combined method of labeling cells with DRAQ5, exposing samples to an antimicrobial agent compared with controls without antimicrobial treatment and measuring differences in fluorescence via flow cytometry as set forth in the rejection of claim 1 above is considered to encompass the measurement of comparative changes in nucleic acid content between exposed and unexposed samples.
Regarding claim 20, Martin teaches the use of DNA staining dyes that provides the capability to assess and compare the size of actively dividing cells as shown in Figure 1A that contains stained cells undergoing mitosis and a scale bar.
Regarding claim 23, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment by 0.05% AgO and 0.05% Clotrimazole. As there is no definition of an inhibitory concentration given by the instant specification, there is the suggestion that “preferably, in the examples and embodiments described herein above, the concentration of the antimicrobial agent is an inhibitory concentration of said antimicrobial agent to which the cells of the microorganism obtained from the subject are susceptible” [para 0043]. Therefore under the broadest reasonable interpretation, an inhibitory concentration of an antimicrobial agent is one to which the microorganism is susceptible. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the concentration of antimicrobial agents disclosed by the method of Madhavan satisfies the limitations of claim 23.
Regarding claim 30, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment with 0.05% AgO and 0.05% Clotrimazole and cell viability below 30% relative to controls after treatment with 0.1% AgO. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the determination of susceptibility to different concentrations of antimicrobial agents disclosed by the method of Madhavan satisfies the limitations of claim 30.
Regarding claim 31, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents at pre-determined concentrations of 0.05% and 0.1% on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment with 0.05% AgO and 0.05% Clotrimazole and cell viability below 30% relative to controls after treatment with 0.1% AgO. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], and as there is no definition of a “pre-determined concentration” in the instant specification or in the claim, under the broadest reasonable interpretation the determination of susceptibility to a concentration of antimicrobial agent that is equal to the pre-determined concentration is disclosed by the method of Madhavan satisfies the limitations of claim 31.
Regarding claims 39 and 124-126, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to the exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of claim 1, namely that cells “were allowed to be treated with a particular formulation for the designated time point (20-180 min)” [p 10, lines 31-32] before being labeled and analyzed by viability assay, and wherein the method does not comprise culturing the cells of the microorganism to identify the microorganism.
Regarding claim 41, the teachings of Madhavan, Martin, Koch and Jorgensen correspond to the steps (i) to (iv) of claim 1 as set forth above. Jorgensen additionally teaches determination of MIC via the broth dilution test method, wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, wherein after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that elicited the observed effect on the cell population which in the case of Jorgensen is inhibition of cell growth, and the lowest concentration of antibiotic that elicited said effect was classified as the MIC [p 1749, col 2, para 1]. As the method of Jorgensen includes multiple dilutions spanning the MIC without explicit bounds, the method is interpreted to include concentrations of antimicrobial that do not elicit the observed effect of cell growth inhibition that would be effectively considered equal to an untreated control sample.
Regarding claim 52, Madhavan, Martin, Jorgensen and Koch teach the steps (i) to (iv) of claim 1 and include measuring cellular morphology via acoustic flow cytometry after treatment of a culture with antimicrobial agents as discussed above and Jorgensen teaches the method of determining MIC as described above, wherein after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that elicited the observed effect on the cell population which in the case of Jorgensen is the inhibition of cell growth [p 1749, col 2, para 1 of Madhavan]. While neither Madhavan, Martin, Jorgensen nor Koch teach the specific range for the determination of the minimal concentration of antimicrobial agent in which the observed effect is equal to or less than 1%-50% of the cells in the control, Jorgensen does teach the determination of the lowest concentration of antibiotic that elicits the observed effect of growth inhibition is considered the MIC. The method for determining MIC of Jorgensen is considered to encompass results that may vary between samples, and therefore inherently involves a comparison to a control sample to determine whether the desired effect is observed that would be judged by one of skill in the art. According to MPEP 2144.05.II.A, where 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. As Madhavan discloses the method of subjecting cells to antimicrobial agents and monitoring changes in cell morphology, and Jorgensen discloses the method of determining MIC by noting the minimum concentration of antimicrobial agent required to elicit the desired effect on the tested culture, one of skill in the art would have been able to use the concepts of Jorgensen with the method of Madhavan to establish the amount of treated cells displaying the desired effect from treatment needed to determine a MIC.
Regarding claim 69, Madhavan discloses a method of culture treatment in Example 1 [p 10, lines 16-26] wherein cells are grown in a culture flask of tryptic soy agar per ATCC guidelines for approximately 30 hours to reach 90% confluence that is incorporated into Example 30 using C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], wherein cells are labeled according to the incorporated Example 2 of using the LIVE/DEAD FungaLight Yeast Viability Kit [p 11, lines 22-23], wherein the kit contents comprise a mixture of SYTO9 and propidium iodide, both of which are nucleic acid binding fluorescence compounds as evidenced by LD [p 1, col 1, para 1] as described above in the rejection of claim 1. As disclosed by the culture protocol of Example 1, “For each experimental time point, a control tube was seeded as well. Sterile paper discs were handled in a biological hood and 1% of the test formulation was carefully smeared therein. The disc was then dropped into the test tube with the broth and placed into the shaking incubator. At the same time, an ‘empty’ disk was dropped into the control broth”, indicating that controls were generated that contained no antimicrobial agent [p 10, lines 24-25], and these control cultures were analyzed alongside samples treated with controls via flow cytometry as outlined by the viability protocol in Example 2.
Regarding claim 123, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract].
Regarding claim 127, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of claim 1, wherein cells are treated with a topical antibiotic formulation including at least one of the panel of antimicrobial agents selected from benzalkonium chloride, benzethonium chloride, salicylic acid, clotrimazole, and miconazole, each of which compounded with AgO as a delivery system [p 1, final paragraph, to p 2, para 1], Jorgensen teaches the use of 96 well plates to conduct tests using approximately 12 antibiotics of 8 two-fold dilutions in a single plate [p 1750, col 1, para 1], and Jorgensen teaches the determination of MIC is an important step to aiding the administration of a therapeutically effective treatment of a patient, and therefore implicitly discloses the administration of a therapeutically effective amount of an agent to which a microorganism has been determined to be susceptible as described in the rejection of claim 1.
Regarding claim 130, Jorgensen teaches the use of 96 well plates to conduct tests using approximately 12 antibiotics of 8 two-fold dilutions in a single plate [p 1750, col 1, para 1].
Regarding claim 131, Jorgensen teaches the broth dilution test method wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, and after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that prevented growth, which was classified as the MIC [p 1749, col 2, para 1].
Regarding claim 132, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] as described above in the rejection of claim 1, wherein the results shown in Figure 9 indicate that the measurements occurred as cells became compromised as evidenced by the decrease over time of live cells relative to the control, which is considered to encompass a measurement before complete cell breakdown, as live cells were still remaining in the sample at the time of data collection.
Regarding claim 136, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract].
Regarding claims 137 and 140, Koch teaches the use of flow cytometry forward scattering date to determine cell volume as stated in the rejection of claim 1, which is considered to correspond to the measurement of cytoplasmic volume.
Regarding claims 138-139, Madhavan discloses the method of determining the percentage of live cells as described in Example 2 [beginning p 10] as a result of the LIVE/DEAD FungaLight Yeast Kit [p 11, lines 22-23], comprising steps of exposure of a labeled sample to an antimicrobial agent compared with controls without antimicrobial treatment [p 11, lines 16-18] that contains SYTO9 and PI as evidenced by LD [p 1, col 1, para 1], and Martin teaches the use of DRAQ5 to label nucleic acid [p 51, col 1, para 3]. The combined method of labeling cells with DRAQ5, exposing samples to an antimicrobial agent compared with controls without antimicrobial treatment and measuring differences in fluorescence via flow cytometry as set forth in the rejection of claim 1 above is considered to encompass the measurement of comparative changes in nucleic acid content between exposed and unexposed samples, and one of skill in the art would recognize such a comparative measurement could be considered distinct from a determination of whether a microorganism is dead or alive.
For these reasons, the invention of claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 11 and 134-135 are rejected under 35 U.S.C. 103 as being unpatentable over Madhavan, Jorgensen, Papich, Martin and Koch as applied to claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 above, and further in view of Assuncao et al. (J Appl Microbiol, 2005, 98:1047; cited on the Form PTO-892 mailed 10/29/2025; herein referred to as Assuncao).
The instant rejection is maintained from a previous office action, and any newly recited portions are necessitated by amendment. The instant rejection is made considering an alternative interpretation of Madhavan wherein the method of Madhavan does not correspond to the limitations of labeling the cells with a single compound and no other compound as recited in claim 1.
Claim 11 is drawn to the method of claim 1, wherein the nucleic acid binding fluorescent compound is a fluorophore dye selected from the group consisting of a cyanine nucleic acid fluorophore dye having an empirical formula C28H27IN2O, cyanine nucleic acid fluorophore dye having an empirical formula C28H27IN2S, Hoechst 33342 having an empirical formula C33H40N5O6, propidium iodine (PI), and 4’,6-diamino-2-phenylindole (DAPI), and any combination thereof.
Claim 134 is drawn to the method of claim 1, wherein the nucleic acid binding fluorescent compound is a cyanine nucleic acid fluorophore dye.
Claim 135 is drawn to the method of claim 1, wherein the microorganism is a bacteria and said method of incubating cells of the microorganism in and/or from the sample in a culture medium for a time and under conditions sufficient for said cells to actively divide comprises incubating said cells for at least 20 minutes, optionally up to 3 hours.
The teachings of Madhavan, Jorgensen, Papich, Martin and Koch as applied to claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 are discussed above. These references do not teach the use of a compound recited in claim 11.
Assuncao relates to the evaluation of Mycoplasma hyopneumoniae growth by flow cytometry [title], and discusses the use of flow cytometry for the rapid, sensitive and specific technique that can be used to analyze a number of parameter that include different aspects of growth and physiology of bacterial populations of different physical sizes [p 1049, col 1, final paragraph].
Regarding claims 11 and 134, Assuncao teaches a method of labeling nucleic acid in M. hyopneumoniae cultures with SYTO9 alone [p 1049, col 2, para 3-5] for analysis via flow cytometry [p 1050, col 1, para 2, exemplary results shown in Figure 3]. As SYTO9 is understood in the art as a cyanine nucleic acid fluorophore and has the empirical formula C28H27IN2O according to the instant specification [para 0029], the SYTO9 of Assuncao corresponds to a cyanine nucleic acid fluorophore and has the empirical formula C28H27IN2O as recited in the claim. As SYTO9 is encompassed by the structural limitations of the nucleic acid binding fluorescent compound of claim 1, it is therefore considered to have the effect of arresting cell division of intact and/or replicating cells of said microorganism, as this function is considered to be inherent to the structure of the nucleic acid binding fluorescent compound (see MPEP 2112.01(I)).
In view of Assuncao, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Madhavan, Jorgensen, Papich, Martin and Koch by using SYTO9 as the sole nucleic acid binding fluorescent compound, as taught by Assuncao, to arrive at the claimed invention, as the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the SYTO9/PI of Madhavan, the DRAQ5 of Martin, and the STYO9 of Assuncao are all nucleic acid binding fluorescent compounds, and as such all are capable of being incorporated into the methods as described by Madhavan, Jorgensen, Papich, Martin and Koch. Thus it would have been obvious to one of ordinary skill in the art to replace the DRAQ5 dye of Martin with the SYTO9 dye of Assuncao as a sole nucleic acid binding fluorescent compound, as one of ordinary skill in the art would have been able to carry out such a substitution with a reasonable expectation of success because both Madhavan, Martin and Assuncao discuss methods of labeling nucleic acids with fluorescent compounds.
Regarding claim 135, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract]. Madhavan additionally teaches analogous antimicrobial agent testing on the bacteria E. coli [Example 4] using a viability assay comprising exposing bacteria to an antimicrobial agent for 20-180 min [Example 2], and Assuncao teaches the labeling of the bacteria M. hyopneumoniae with SYTO9 for the assessment of growth via flow cytometry. One of skill in the art would therefore be able to apply the combined method of Madhavan, Jorgensen, Papich, Martin, and Koch to a bacterium using the fluorescent compound of Assuncao, which would correspond to the limitations of claim 135.
Therefore, the invention of claims 11 and 134-135 would have been obvious to one of ordinary skill in the art before the effective filing date.
Response to Remarks: Beginning on page 17 of Applicant’s response to rejections under 35 USC 103; Applicant in summary contends there is no clearly-articulated reason why one of ordinary skill in the art would look to the cited references to make the proposed modifications; Applicant further contends the prior art of record do not disclose the use of the change in cell volume to determine the effect of an antimicrobial agent, and do not disclose the correlation between cell volume and susceptibility of a microorganism to an antimicrobial agent; Applicant further contends there is no reason why one of ordinary skill in thee art would look to Koch to modify the method of Madhavan; Applicant further contends the there is no reason to modify the use of labels as taught by Madhavan for using flow cytometry size determinations as taught by Koch to achieve the same outcome, as Koch is not drawn to studying the effects of antimicrobials on a cell; Applicant further contends hindsight reconstruction in view of Applicant’s present disclosure is the only reason one of skill in the art would look to Koch.
Applicant’s remarks are considered and found not convincing.
Regarding the motivations for why one of skill in the art would modify the method of Madhavan with the prior art elements, these motivations are set forth in the rejection above, and are summarized here:
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by determining MIC of the tested antimicrobial agent, because Jorgensen teaches that determining MIC provides a qualitative and quantitative assessment of whether an organism is susceptible, intermediate, or resistant to an antimicrobial agent and such determinations can aid a physician in selecting a particular drug for an effective therapy against infection.
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by assessing the cell volumes and sizes via the collected flow cytometry data, because Madhavan teaches a method of assessing growth characteristics of cells treated with antimicrobial agents via flow cytometry, and Koch teaches using flow cytometry data to assess cellular volumes and sizes is an important variable in studies involving kinetic models of growth, nutrient transport and metabolism.
One of ordinary skill in the art would have been motivated to modify the method of Madhavan by administering a therapeutically effective amount of the antimicrobial agent to a subject, because Papich discloses that administration must ensure there is sufficient drug to eliminate susceptible isolates achieved by maintaining drug concentrations in serum above the MIC for a portion of the dose interval, and gives examples of treating gram-negative infection with regimens of penicillins and cephalosporins for 3-4 times per day.
Regarding the assertion that the prior art of record do not disclose the use of the change in cell volume to determine the effect of an antimicrobial agent, and do not disclose the correlation between cell volume and susceptibility of a microorganism to an antimicrobial agent, and the assertion that there is no reason to modify the use of labels as taught by Madhavan for using flow cytometry size determinations as taught by Koch to achieve the same outcome, as Koch is not drawn to studying the effects of antimicrobials on a cell:
Madhavan teaches a method of determining susceptibility of a cell to antimicrobial agents comprising labeling of cells and flow cytometry, and Koch discloses the use of flow cytometry data to assess cellular volumes and sizes is an important variable in studies involving kinetic models of growth. The assessment of susceptibility of a cell to an antimicrobial agent as disclosed by Madhavan is a study involving the growth of a cell, as the user is observing cells and their ability to grow in the presence of said antimicrobial agent via collected flow cytometry data. The method of Madhavan additionally uses the same data which Koch suggests is useful for assessing cellular volumes and sizes to study growth. Therefore one of skill in the art would be motivated to carry out the method of Madhavan and additionally use the subsequent flow cytometry data to assess cellular volumes and size as taught by Koch, as Koch considers these variables to be important for studies involving growth.
While Koch does not teach a correlation between cell volumes and antimicrobial agent susceptibility, the claims do not exclusively limit the determination of susceptibility of a sample to an antimicrobial agent in the claimed method to solely assessing changes in cell volume in view of the phrase “wherein said measuring comprises”, which indicates other steps and types of measuring may be included. Put another way, the recitation “wherein said measuring comprises” in step (iv) of claim 1 does not exclusively limit the measurements to be measurements of cell morphology and changes in size and/or volume of cytoplasmic volume. Additionally claim 1 step (v) recites that the determination of susceptibility is “from the acoustic flow cytometry data output”, which does not exclusively limit the method to establish any correlation between cell volumes and antimicrobial agent susceptibility nor to use any of the resulting relationships that may arise in the determination of susceptibility.
In response to applicant’s argument that the examiner’s conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Double Patenting
Claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,072,274 (herein “patent”; issued from application 17/276717) in view of Madhavan, Jorgensen, Martin, Papich and Koch and as evidenced by LD, ATCC, ATTUNE and Dabrowa.
The instant rejection is maintained from a previous office action, and any newly recited portions are necessitated by amendment.
Regarding instant claim 1, claim 1 of the patent recites a method of antimicrobial agent susceptibility testing comprising preparing microorganism samples in an electrolyte that are either exposed or not exposed to antimicrobials, measuring a signal in an impedance flow cytometer, comparing signals between exposed samples and nonexposed controls, and determining the susceptibility to the microbial agent based on the comparison of signals.
The claim(s) of the patent do(es) not recite the limitations related to incubating cells in culture media, exposing cells to an antimicrobial agent for a time and under conditions sufficient for said cells of the microorganism to actively divide, labelling cells, the measuring effect of said antimicrobial agent on cellular morphology of actively dividing cells of the microorganism by acoustic flow cytometry, the determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, and the treatment of a subject by administering a therapeutically effective amount of the antimicrobial agent.
Madhavan describes antimicrobial formulations [title] that are suitable for use against bacterial and fungal organisms [p 9, lines 25-27], .
Regarding instant claim 1 and the limitations of step (i) of incubating in a culture medium cells of the microorganism from a sample obtained from a subject having or suspected of having an infection with said microorganism, Madhavan discloses the process of culture treatment in Example 1 [p 10, lines 16-26] wherein cells are grown in a culture flask of tryptic soy agar per ATCC guidelines for approximately 30 hours to reach 90% confluence. Madhavan similarly applies the method of Example 1 in Example 30 [p 21, lines 8-14] to the fungal organism Candida albicans ATCC 10231 that is a microorganism isolated from a man with bronchomycosis as evidenced by ATCC [p 1, col 1].
Regarding instant claim 1 and the limitations of step (ii) of exposing cells of the microorganism and/or from the actively dividing cell culture obtained in step (i) to an antimicrobial agent for a time and under conditions sufficient for said cells of the microorganism to actively divide, Madhavan discloses treating the fungal sample with a formulation for 20-180 min along with a corresponding control [p 11, lines 16-18], which is considered to correspond to a time and under conditions sufficient for said cells of the microorganism to actively divide. Additionally, the corresponding control of Madhavan is considered to correspond to a sample incubated in parallel without exposure to the antimicrobial agent.
Regarding instant claim 1 and the limitations of step (iii) of labelling cells of the microorganism exposed to the antimicrobial agent in step (ii) with a nucleic acid binding fluorescent compound, Madhavan discloses the step of adding contents of the LIVE/DEAD FungaLight Yeast Viability Kit to the fungal cells after exposure to antimicrobial formulation as described in the viability assay protocol of Example 2 [p 11, lines 22-23], wherein the kit contents comprise a mixture of SYTO9 and propidium iodide, both of which are nucleic acid binding fluorescence compounds as evidenced by LD [p 1, col 1, para 1]. As Madhavan discloses “adding 10 ul of the LIVE/DEAD Funga Light Yeast Viability Kit” to the cells and incubated for 5 minutes [Example 2, p 11, lines 22-23], and the kit comprises both SYTO9 and PI with instructions to add compounds both singularly and together to samples as evidenced by LD [p 2, col 2, sections 2.2-2.4], the method of Madhavan is considered to encompass both the addition of both SYTO9 and PI together to samples, as well as SYTO9 and PI individually to separate samples. Therefore, the teachings of Madhavan extend to the labeling of cells by a single nucleic acid binding fluorescent compound, as live cells would only be labeled by SYTO9 considering that PI only interacts with dead cells. Additionally, as the SYTO9 of Madhavan is encompassed by the structural limitations of a nucleic acid binding fluorescent compound set forth in the claim, it is presumed to have the activity of being capable of arresting cell division, as the activity of the compound is presumed to be inherent to its structure (see MPEP 2112.01.I). Additionally, the limitation of “wherein said method does not comprise labeling the cells of the microorganism with any other compound”, the term “the cells” can be broadly interpreted to encompass all of the cells of the microorganism in the sample. In view of this interpretation, as the live cells of Madhavan are only labeled by SYTO9, all of the cells of the microorganism in the sample are not labeled with any other compound.
Regarding instant claim 1 and the limitations in step (iv) of measuring effect of said antimicrobial agent on cellular morphology of said cells of the microorganism by acoustic flow cytometry, Madhavan discloses the analysis of labeled samples using the Attune flow cytometer [p 10, lines 7-8], which is an acoustic flow cytometer as evidenced by ATTUNE [p 1, title] to mathematically determine the number of cells as a percentage of the control [p 11, lines 24-26].
Regarding instant claim 1 and the limitations in step (v)(A) of determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, comprising determining susceptibility of the microorganism to the antimicrobial agent qualitatively by determining and/or quantifying from the acoustic flow cytometry data output that said microorganism is either susceptible or not susceptible to said antimicrobial agent, Madhavan discloses the analysis of labeled samples using the Attune flow cytometer [p 10, lines 7-8], which is an acoustic flow cytometer as evidenced by ATTUNE [p 1, title] to mathematically determine the number of cells as a percentage of the control [p 11, lines 24-26]. As SYTO9 is used to label all yeast cells including both cells with intact membranes and those with damaged membranes, and PI is used to label only cells with damaged membranes as evidenced by LD, the comparison of these labeled samples to controls is effectively a measurement of the change in cell morphology as it relates to the damage of cell membranes by the antimicrobial agent. Given the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the method disclosed by Madhavan satisfies this limitation of instant claim 1.
Jorgensen discusses the principles of antimicrobial susceptibility testing [title], wherein MIC is discussed as the lowest concentration of antibiotic that prevents growth [p 1, col 2, para 1], and that the determination of the MIC provides a quantitative and qualitative assessments of whether an organism is susceptible, intermediate, or resistant to an antimicrobial [abstract].
Regarding instant claim 1 and the limitations in step (v)(B) of determining and/or quantifying susceptibility of the microorganism to the antimicrobial agent from the acoustic flow cytometry data output, comprising determining susceptibility of the microorganism to the antimicrobial agent quantitatively by determining and/or quantifying from the acoustic flow cytometry data output the minimal inhibitory concentration (MIC) of said antimicrobial agent to which the microorganism is susceptible, Jorgensen discloses the broth dilution test method wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, and after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that prevented growth, which was classified as the MIC [p 1749, col 2, para 1]. As Madhavan discloses the use of acoustic flow cytometry methods to gather data related to the effect of antimicrobial agents on cells through the comparison of nucleic acid amounts of exposed cells to that of unexposed cells, and Jorgensen teaches the method for determining MIC using data related to cell survivability as a result of exposure to different concentrations of antimicrobial agent, one of skill in the art would be capable of applying the data produced by the method of Madhavan to determine MIC based on the concepts disclosed by Jorgensen.
Regarding instant claim 1 and the limitations in step (vi) of treating a subject having or suspected of having an infection with the unicellular or multicellular microorganism by administering to the subject a therapeutically effective amount of said antimicrobial agent, Jorgensen discloses the results of a susceptibility test such as MIC must be interpreted and reported to a physician, which includes interpretation of the likely success of a particular agent in eradicating bacteria at various body sites [p 1753, col 1, para 4], and that determination of MIC and classification of organisms as resistant, susceptible, or intermediate aid a physician in selecting a particular drug for an effective therapy [p 1753, col 2, para 2]. Therefore Jorgensen discloses the determination of MIC is an important step to aiding the administration of a therapeutically effective treatment of a patient, and therefore implicitly discloses the administration of a therapeutically effective amount of an agent to which a microorganism has been determined to be susceptible.
Considering an alternative interpretation of Jorgensen, wherein Jorgensen does not implicitly disclose the administration of a therapeutically effective amount of an antimicrobial agent, Papich discusses the rational selection of dosage regimes for the prudent use of antimicrobial drugs [title], wherein it is taught that administration must ensure there is sufficient drug to eliminate susceptible isolates [p 480, col 1, para 1].
Regarding instant claim 1 and the limitations in step (vi), Papich discloses administration must ensure there is sufficient drug to eliminate susceptible isolates [p 480, col 1, para 1], which is achieved by maintaining drug concentrations in serum above the MIC for a portion of the dose interval [p 482, col 1, para 3], which is considered to encompass a therapeutically effective amount of the antimicrobial agent. Papich further provides examples that for treating of a gram-negative infection, especially a serious one, some regimens for penicillins and cephalosporins require administration 3-4 times per day [beginning p 482, col 2, para 5], and that cephalexin and amoxicillin-clavulanate have been used to successfully treat staphylococcal infections when administers once daily.
Considering an alternative interpretation wherein the method of Madhavan is considered not to correspond to the limitations of labeling the cells with a single compound and no other compound, Martin discusses DNA labeling in living cells [title], wherein different fluorescent dyes are discussed in terms of their advantages with different biological applications, such as the use of DRAQ5 for its ability to stain DNA in living cells with low photobleaching and deep red excitation/emission [abstract].
Regarding instant claim 1 and the limitations in step (iii) of labeling cells with a single nucleic acid dye that results in arrest of cell division and labeling the cells with no other compound, Martin discloses the labeling of cells by staining DNA with DRAQ5 fluorescent dye can hinder cell cycle progression at the G2 phase because it is an anthracycline derivative that intercalates into DNA and conceivably blocks topoisomerase II [p 51, col 1, para 3]. As the method of Madhavan cited above is carried out on the yeast microorganism C. albicans, one of skill in the art would recognize that DRAQ5 would be applicable to arresting cell development in this organism at G2 phase due to its intercalation activity.
Koch relates to the deduction of cell volume and mass for forward scatter intensity of bacteria analyzed by flow cytometry [title], and describes the theory that forward scatter signals from a flow cytometer can be converted to cellular volumes that are applicable to bacteria [abstract], which is an important variable in studies involving kinetic models of growth, nutrient transport and metabolism.
Regarding instant claim 1 and the limitations in step (vi) of measuring an increase in size and/or cytoplasmic volume of actively dividing cells, Koch discloses a method of using flow cytometry measurements of forward scattering intensity of bacterial samples to calculate cell volume, and teaches that such a method is built on the relationship between forward scatter intensity and cell size [p 50, col 1, para 1]. Koch indicates the distinction of this method is in part due to the size of bacteria being substantially smaller than mammalian cells that were more frequently studied by flow cytometry at the time of the references publication [p 50, col 2, para 1], therefore implying that the assessment of cell volume via flow cytometry was routine for cells larger than bacteria at that time via forward scattering analysis, and that the method presented in the reference made available the application of cell volume measurements on bacteria. Therefore the disclosure of Koch involving the use of flow cytometry forward scattering data to assess cell volume are considered to correspond to the limitation of measuring an increase in size and/or cytoplasmic volume of actively dividing cells.
In view of Madhavan, Jorgensen, Papich and Koch, it would have obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by culturing cells, labeling cells, exposing cells to antimicrobial agents and analyzing the cultures via acoustic flow cytometry as disclosed by Madhavan, by determining MIC and administering a therapeutically effective amount of an antimicrobial agent, as disclosed by Jorgensen, administering the antimicrobial agent to a subject with infection, as disclosed by Papich, and measuring an increase in cell size and/or cytoplasmic volume, as disclosed by Koch, to arrive at the claimed invention.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by using acoustic flow cytometry, because Madhavan discloses methods and antimicrobial formulations suitable for use against bacterial and fungal organisms as determined by acoustic flow cytometry.
One of ordinary skill in the art would have been motivated to modify the claims of the patent to determine the MIC of a tested antimicrobial agent because Jorgensen discloses that determining MIC provides a qualitative and quantitative assessment of whether an organism is susceptible, intermediate, or resistant to an antimicrobial agent and such determinations can aid a physician in selecting a particular drug for an effective therapy against infection.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by measuring the cell volume and size of the organisms using the flow cytometry data because Koch discloses using flow cytometry data to assess cellular volumes and sizes is an important variable in studies involving kinetic models of growth, nutrient transport and metabolism.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by administering a therapeutically effective amount of the tested antimicrobial agents to a subject because Papich discloses that administration must ensure there is sufficient drug to eliminate susceptible isolates achieved by maintaining drug concentrations in serum above the MIC for a portion of the dose interval, and gives examples of treating gram-negative infection with regimens of penicillins and cephalosporins for 3-4 times per day.
One of ordinary skill in the art would have had a reasonable expectation of success because both the patent and Madhavan relate to methods for antimicrobial agent susceptibility testing involving flow cytometry, Madhavan and Jorgensen discuss techniques for determining the susceptibility of microorganisms to antimicrobial agents, Madhavan and Koch discuss techniques for using flow cytometry data to assess characteristics of cells throughout growth, and Jorgensen and Papich discuss the administration of sufficient amounts of antimicrobials to treat microbial infection as an application of determined antimicrobial susceptibility data.
In view of Martin, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of the patent, Madhavan, Jorgensen, Papich and Koch by using DRAQ5 as a nucleic acid binding fluorescent compound, as disclosed by Martin, to arrive at the claimed invention, as the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the SYTO9 and PI of Madhavan and the DRAQ5 of Martin are nucleic acid binding fluorescent compounds, and as such all are capable of being incorporated into such methods as described by Madhavan. Thus it would have been obvious to one of ordinary skill in the art to replace the SYTO9 and PI dyes of Madhavan with the DRAQ5 dye of Martin, as one of ordinary skill in the art would have been able to carry out such a substitution with a reasonable expectation of success because both Madhavan and Martin discuss methods of labeling nucleic acids with fluorescent compounds.
Regarding instant claim 17, Madhavan discloses the method of determining the percentage of live cells as described in Example 2 [beginning p 10] as a result of the LIVE/DEAD FungaLight Yeast Kit [p 11, lines 22-23] comprising steps of exposure of a fluorescently labeled sample to an antimicrobial agent compared with controls without antimicrobial treatment [p 11, lines 16-18] that contains SYTO9 and PI as evidenced by LD [p 1, col 1, para 1], and Martin discloses the use of DRAQ5 to label nucleic acid [p 51, col 1, para 3]. The combined method of labeling cells with DRAQ5, exposing samples to an antimicrobial agent compared with controls without antimicrobial treatment and measuring differences in fluorescence via flow cytometry as set forth in the rejection of instant claim 1 above is considered to encompass the measurement of comparative changes in nucleic acid content between exposed and unexposed samples.
Regarding instant claim 20, Martin discloses the use of DNA staining dyes that provides the capability to assess and compare the size of actively dividing cells as shown in Figure 1A that contains stained cells undergoing mitosis and a scale bar.
Regarding instant claim 23, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of instant claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment by 0.05% AgO and 0.05% Clotrimazole. As there is no definition of an inhibitory concentration given by the instant specification, there is the suggestion that “preferably, in the examples and embodiments described herein above, the concentration of the antimicrobial agent is an inhibitory concentration of said antimicrobial agent to which the cells of the microorganism obtained from the subject are susceptible” [para 0043]. Therefore under the broadest reasonable interpretation, an inhibitory concentration of an antimicrobial agent is one to which the microorganism is susceptible. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the concentration of antimicrobial agents disclosed by the method of Madhavan satisfies the limitations of instant claim 23.
Regarding instant claim 30, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of instant claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment with 0.05% AgO and 0.05% Clotrimazole and cell viability below 30% relative to controls after treatment with 0.1% AgO. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], the determination of susceptibility to different concentrations of antimicrobial agents disclosed by the method of Madhavan satisfies the limitations of instant claim 30.
Regarding instant claim 31, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents at pre-determined concentrations of 0.05% and 0.1% on C. albicans according to exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of instant claim 1, wherein Figure 9 shows a reduction of cell viability below 10% relative to untreated controls in 20 minutes after treatment with 0.05% AgO and 0.05% Clotrimazole and cell viability below 30% relative to controls after treatment with 0.1% AgO. As the phrase “susceptibility of the microorganism to antimicrobial agent(s)” is defined as “the one or more antimicrobial agents promote damage to, or compromise the integrity of, the cellular morphology of actively dividing cells of the microorganism … when exposed to, or cultured with an amount of the antimicrobial agent compared with the …. cells of the microorganism … cultured in the absence of the antimicrobial agent” in the instant specification [para 00245], and as there is no definition of a “pre-determined concentration” in the instant specification or in the instant claim, under the broadest reasonable interpretation the determination of susceptibility to a concentration of antimicrobial agent that is equal to the pre-determined concentration is disclosed by the method of Madhavan satisfies the limitations of instant claim 31.
Regarding instant claims 39 and 124-126, Madhavan discloses a method in Example 30 [p 21, lines 8-14 and Figure 9] of determining the effect of antimicrobial agents on C. albicans according to the exposure and viability protocols described in Examples 1 and 2, respectively, as described above in the rejection of instant claim 1, namely that cells “were allowed to be treated with a particular formulation for the designated time point (20-180 min)” [p 10, lines 31-32] before being labeled and analyzed by viability assay, and wherein the method does not comprise culturing the cells of the microorganism to identify the microorganism.
Regarding instant claim 41, the disclosures of Madhavan, Martin, Koch and Jorgensen correspond to the steps (i) to (iv) of instant claim 1 as set forth above. Jorgensen additionally teaches determination of MIC via the broth dilution test method, wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, wherein after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that elicited the observed effect on the cell population which in the case of Jorgensen is inhibition of cell growth, and the lowest concentration of antibiotic that elicited said effect was classified as the MIC [p 1749, col 2, para 1]. As the method of Jorgensen includes multiple dilutions spanning the MIC without explicit bounds, the method is interpreted to include concentrations of antimicrobial that do not elicit the observed effect of cell growth inhibition that would be effectively considered equal to an untreated control sample.
Regarding instant claim 52, Madhavan, Martin, Jorgensen and Koch teach steps (i) to (iv) of instant claim 1 as discussed above, and include measuring cellular morphology via acoustic flow cytometry after treatment of a culture with antimicrobial agents as discussed above and Jorgensen teaches the method of determining MIC as described above, wherein after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that elicited the observed effect on the cell population which in the case of Jorgensen is the inhibition of cell growth [p 1749, col 2, para 1 of Madhavan]. While neither Madhavan, Martin, Jorgensen nor Koch disclose the specific range for the determination of the minimal concentration of antimicrobial agent in which the observed effect is equal to or less than 1%-50% of the cells in the control, Jorgensen does disclose the determination of the lowest concentration of antibiotic that elicits the observed effect of growth inhibition is considered the MIC. The method for determining MIC of Jorgensen is considered to encompass results that may vary between samples, and therefore inherently involves a comparison to a control sample to determine whether the desired effect is observed that would be judged by one of skill in the art. According to MPEP 2144.05.II.A, where 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. As Madhavan discloses the method of subjecting cells to antimicrobial agents and monitoring changes in cell morphology, and Jorgensen discloses the method of determining MIC by noting the minimum concentration of antimicrobial agent required to elicit the desired effect on the tested culture, one of skill in the art would have been able to use the concepts of Jorgensen with the method of Madhavan to establish the amount of treated cells displaying the desired effect from treatment needed to determine a MIC.
Regarding instant claim 69, Madhavan discloses a method of culture treatment in Example 1 [p 10, lines 16-26] wherein cells are grown in a culture flask of tryptic soy agar per ATCC guidelines for approximately 30 hours to reach 90% confluence that is incorporated into Example 30 using C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], wherein cells are labeled according to the incorporated Example 2 of using the LIVE/DEAD FungaLight Yeast Viability Kit [p 11, lines 22-23], wherein the kit contents comprise a mixture of SYTO9 and propidium iodide, both of which are nucleic acid binding fluorescence compounds as evidenced by LD [p 1, col 1, para 1] as described above in the rejection of instant claim 1. As disclosed by the culture protocol of Example 1, “For each experimental time point, a control tube was seeded as well. Sterile paper discs were handled in a biological hood and 1% of the test formulation was carefully smeared therein. The disc was then dropped into the test tube with the broth and placed into the shaking incubator. At the same time, an ‘empty’ disk was dropped into the control broth”, indicating that controls were generated that contained no antimicrobial agent [p 10, lines 24-25], and these control cultures were analyzed alongside samples treated with controls via flow cytometry as outlined by the viability protocol in Example 2.
Regarding instant claim 123, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of instant claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract].
Regarding instant claim 127, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of instant claim 1, wherein cells are treated with a topical antibiotic formulation including at least one of the panel of antimicrobial agents selected from benzalkonium chloride, benzethonium chloride, salicylic acid, clotrimazole, and miconazole, each of which compounded with AgO as a delivery system [p 1, final paragraph, to p 2, para 1], Jorgensen discloses the use of 96 well plates to conduct tests using approximately 12 antibiotics of 8 two-fold dilutions in a single plate [p 1750, col 1, para 1], and Jorgensen teaches the determination of MIC is an important step to aiding the administration of a therapeutically effective treatment of a patient, and therefore implicitly discloses the administration of a therapeutically effective amount of an agent to which a microorganism has been determined to be susceptible as described in the rejection of instant claim 1.
Regarding instant claim 130, Jorgensen discloses the use of 96 well plates to conduct tests using approximately 12 antibiotics of 8 two-fold dilutions in a single plate [p 1750, col 1, para 1].
Regarding instant claim 131, Jorgensen discloses the broth dilution test method wherein two-fold dilutions of antibiotics are prepared in a liquid growth medium in test tubes, and then inoculated with standardized bacterial suspensions of 1-5 x 105 CFU/ml, and after overnight incubation at 35 °C tubes are examined for turbidity and determination of the lowest concentration of antibiotic that prevented growth, which was classified as the MIC [p 1749, col 2, para 1].
Regarding instant claim 132, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] as described above in the rejection of instant claim 1, wherein the results shown in Figure 9 indicate that the measurements occurred as cells became compromised as evidenced by the decrease over time of live cells relative to the control, which is considered to encompass a measurement before complete cell breakdown, as live cells were still remaining in the sample at the time of data collection.
Regarding instant claim 136, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of instant claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract].
Regarding instant claims 137 and 140, Koch discloses the use of flow cytometry forward scattering date to determine cell volume as stated in the rejection of instant claim 1, which is considered to correspond to the measurement of cytoplasmic volume.
Regarding instant claims 138-139, Madhavan discloses the method of determining the percentage of live cells as described in Example 2 [beginning p 10] as a result of the LIVE/DEAD FungaLight Yeast Kit [p 11, lines 22-23], comprising steps of exposure of a labeled sample to an antimicrobial agent compared with controls without antimicrobial treatment [p 11, lines 16-18] that contains SYTO9 and PI as evidenced by LD [p 1, col 1, para 1], and Martin discloses the use of DRAQ5 to label nucleic acid [p 51, col 1, para 3]. The combined method of labeling cells with DRAQ5, exposing samples to an antimicrobial agent compared with controls without antimicrobial treatment and measuring differences in fluorescence via flow cytometry as set forth in the rejection of instant claim 1 above is considered to encompass the measurement of comparative changes in nucleic acid content between exposed and unexposed samples, and one of skill in the art would recognize such a comparative measurement could be considered distinct from a determination of whether a microorganism is dead or alive.
Claims 11 and 134-135 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,072,274 in view of Madhavan, Jorgensen, Martin, Papich and Koch as applied to claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 above, and further in view of Assuncao.
The instant rejection is maintained from a previous office action, and any newly recited portions are necessitated by amendment. The instant rejection is made considering an alternative interpretation of Madhavan wherein the method of Madhavan does not correspond to the limitations of labeling the cells with a single compound and no other compound as recited in instant claim 1.
The claims of the patent and the disclosures of Madhavan, Jorgensen, Papich, Martin and Koch as applied to instant claims 1, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 136-140 are discussed above. The claims of the patent do not recite the use of compound recited in instant claim 11.
Assuncao relates to the evaluation of Mycoplasma hyopneumoniae growth by flow cytometry [title], and discusses the use of flow cytometry for the rapid, sensitive and specific technique that can be used to analyze a number of parameter that include different aspects of growth and physiology of bacterial populations of different physical sizes [p 1049, col 1, final paragraph].
Regarding instant claims 11 and 134, Assuncao discloses a method of labeling nucleic acid in M. hyopneumoniae cultures with SYTO9 alone [p 1049, col 2, para 3-5] for analysis via flow cytometry [p 1050, col 1, para 2, exemplary results shown in Figure 3]. As SYTO9 is understood in the art as a cyanine nucleic acid fluorophore and has the empirical formula C28H27IN2O according to the instant specification [para 0029], the SYTO9 of Assuncao corresponds to a cyanine nucleic acid fluorophore and has the empirical formula C28H27IN2O as recited in the instant claim. As SYTO9 is encompassed by the structural limitations of the nucleic acid binding fluorescent compound of instant claim 1, it is therefore considered to have the effect of arresting cell division of intact and/or replicating cells of said microorganism, as this function is considered to be inherent to the structure of the nucleic acid binding fluorescent compound (see MPEP 2112.01(I)).
In view of Assuncao, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of the patent, Madhavan, Jorgensen, Papich, Martin and Koch by using SYTO9 as the sole nucleic acid binding fluorescent compound, as disclosed by Assuncao, to arrive at the claimed invention, as the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the SYTO9/PI of Madhavan, the DRAQ5 of Martin, and the STYO9 of Assuncao are all nucleic acid binding fluorescent compounds, and as such all are capable of being incorporated into the methods as described by Madhavan, Jorgensen, Papich, Martin and Koch. Thus it would have been obvious to one of ordinary skill in the art to replace the DRAQ5 dye of Martin with the SYTO9 dye of Assuncao as a sole nucleic acid binding fluorescent compound, as one of ordinary skill in the art would have been able to carry out such a substitution with a reasonable expectation of success because both Madhavan, Martin and Assuncao discuss methods of labeling nucleic acids with fluorescent compounds.
Regarding instant claim 135, Madhavan discloses a method in Example 30 for testing antimicrobial agents on C. albicans ATCC 10231 [p 21, lines 8-14 and Figure 9], that incorporates the culturing protocols outlined in Example 1 [p 9, lines 16-26] and viability assay protocol outlined in Example 2 [beginning p 10] as described above in the rejection of instant claim 1, wherein C. albicans ATCC 10231 is exposed to an antimicrobial agent for 20-180 min [p 11, lines 16-18], allowing for up to 1.7 generations based on the generation time of C. albicans in Saboraud growth medium as evidenced by Dabrowa [abstract]. Madhavan additionally discloses analogous antimicrobial agent testing on the bacteria E. coli [Example 4] using a viability assay comprising exposing bacteria to an antimicrobial agent for 20-180 min [Example 2], and Assuncao discloses the labeling of the bacteria M. hyopneumoniae with SYTO9 for the assessment of growth via flow cytometry. One of skill in the art would therefore be able to apply the combined method of Madhavan, Jorgensen, Papich, Martin, and Koch to a bacterium using the fluorescent compound of Assuncao, corresponding to the limitations of instant claim 135.
Response to Remarks: Beginning on page 23 of Applicant’s response to double patenting rejections; Applicant in summary contends the conflicting patent does not recite or teach the use of acoustic flow cytometry, as the conflicting patent is drawn to the use of impedance flow cytometry which does not measure effects on cells by optical measurements.
Applicant’s remarks are considered and found not convincing. As claim 1 of the patent recites a method of antimicrobial agent susceptibility testing of a microorganism, and the amended instant claim 1 recites a method for determining and/or quantifying susceptibility of a unicellular or multicellular organism, the two are considered to have overlapping subject matter. The details of the rejection such as the motivations for why one of skill in the art would modify the claims of the patent with different prior art elements to arrive at the claimed invention are set forth above.
Conclusion
Status of the Claims:
Claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 are pending.
Claims 1, 11, 17, 20, 23, 30-31, 39, 41, 52, 69, 123-127, 130-132 and 134-140 are rejected.
No claim is in condition for allowance.
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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/JOSEPH R SPANGLER/
Examiner
Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
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