Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Aug. 4, 2023. Claims 1-12 are pending and currently examined.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-5 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Base claim 1 recites the term “suitable” in several limitations. E.g., it recites “wherein each of said plurality of wells is suitable for containing one of a plurality of phages, wherein said microwell plate is further suitable for having a sample comprising said bacterium dispensed into each of said plurality of wells”. The term suitable is a relative term which renders the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102 of this title, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-12 are rejected under 35 U.S.C. 103 as being unpatentable over Xie et al. (Viruses 2018, 10, 189; submitted in IDS filed on Aug. 4, 2023), Henry et al. (Bacteriophage, 2012, 2:3, 159–167), Fischer et al. (PLoS ONE, 2013, 8(1): e53899; submitted in IDS filed on Aug. 4, 2023), and Paczesny et al. (Viruses 2020, 12, 845; submitted in IDS filed on Aug. 4, 2023).
Claims 1-5 are directed to a system for measuring the sensitivity of a bacterium to a plurality of phages, the system comprising:
(1) a microwell plate comprising a plurality of wells, wherein each of said plurality of wells is suitable for containing one of a plurality of phages, wherein said microwell plate is further suitable for having a sample comprising said bacterium dispensed into each of said plurality of wells;
(2) at least one reagent or additive, suitable for being added to at least one mixture of said sample comprising said bacterium with a phage of said plurality of phages; and
(3) a control unit configured to interpret and present the results of measurements in the form of a phagogram, said measurements being provided by at least one analytical device, said at least one analytical device being configured to measure the interaction of each of said plurality of phages with said bacterium;
wherein said at least one reagent and/or additive comprises at least one of the following: a primer molecule, a DNA polymerase, a nucleoside triphosphate (dNTP), a molecular beacon probe capable to fluoresce, a buffer, and a qPCR-amplification compatible solvent and/or a qPCR-amplification compatible enzyme; and
wherein said at least one analytical device is further configured to measure the interaction of each of said plurality of phages with said bacterium by measuring at least one of the abundance and/or differential concentration of phage DNA by use of quantitative polymerase chain reaction; or,
wherein said at least one reagent and/or additive comprises at least one of the following: a Luciferin/Luciferase complex configured to allow detection of bacterial cell lysis through depletion of ATP and concomitant fluorescein light emission, and a Luciferin/Luciferase complex compatible solvent, and/or a Luciferin/Luciferase complex compatible enzyme, and
wherein said at least one analytical device is further configured to measure the interaction of each of said plurality of phages with said bacterium by measuring said light emission.
Claim 6-11 are directed to a method for measuring the sensitivity of a bacterium to a plurality of phages, the method having the following steps:
(1) providing a first composition comprising said bacterium and a first microwell plate comprising a plurality of wells, each of said plurality of wells containing one of said plurality of phages;
(2) dispensing a first sample of said first composition in at least one well of said plurality of wells; for each of said at least one well, allowing incubation of the a mixture of said first sample comprising said bacterium and a bacteriophage contained in the well for the duration of a first period of time;
(3) adding at least one reagent or additive to each mixture;
(4) transporting to and introducing at least one mixture in an analytical device and measuring the bacterium-phage interaction of said at least one mixture by said analytical device; and
(5) interpreting and presenting the measurements by said analytical device in the form of a phagogram by a control unit;
wherein said at least one reagent and/or additive comprises at least one of the following: a primer molecule, a DNA polymerase, a nucleoside triphosphate (dNTP), a molecular beacon probe capable to fluoresce, a buffer, and a qPCR-amplification compatible solvent, and/or a qPCR-amplification compatible enzyme; and
wherein said at least one analytical device is further configured to measure the interaction of each of said plurality of phages with said bacterium by measuring at least one of the abundance and/or differential concentration of phage DNA by use of quantitative polymerase chain reaction;
or,
wherein said at least one reagent and/or additive comprises at least one of the following: a Luciferin/Luciferase complex configured to allow detection of bacterial cell lysis through depletion of ATP and concomitant fluorescein light emission, and a Luciferin/Luciferase complex compatible solvent, and/or a Luciferin / Luciferase complex compatible enzyme, and
wherein said at least one analytical device is further configured to measure the interaction of each of said plurality of phages with said bacterium by measuring said light emission.
Claim 12 is directed to a non-transitory computer program product comprising code configured to cause a processor of a control unit to perform the steps of claim 6.
The Specification teaches that the term "Phagogram" refers to a collection of data, preferably in the form of a table, summarizing the effect of a plurality of individual bacteriophages on either an individual bacterial pathogen or mixed sample environment; that, in some respects equivalent to an antibiogram, the phagogram gives a profile of an organism's resistance or susceptibility to a panel of phages. See [0064].
Xie teaches a study on development and validation of a microtiter plate-based assay for determination of bacteriophage host range and virulence. Xie teaches that the most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format. See Abstract.
Specifically, Xie teaches, for the microtiter plate host range assay, that A subset of four bacterial strains (S. Anatum strain FC1033C3, S. Newport strain USDA2, S. Typhimurium strain USDA1, and S. Enteritidis strain SGSC 2475) and four phages (Sasha, Season12, Munch, and Sw2) were selected to develop the parameters for the microtiter plate liquid-culture host range assay. Different initial bacterial inoculum levels were tested in combination with phages at starting concentrations of 106 to 108 PFU/mL. The low inoculum condition (~105 CFU/mL) was achieved by adjusting fresh overnight cultures OD550nm ~0.5 and diluting 1000-fold in TSB. For the high inoculum condition, fresh overnight cultures were adjusted with TSB to OD550nm ~0.1 to achieve a concentration of ~108 CFU/mL. Phage lysates were titered and adjusted to concentrations of 107, 108, and 109 PFU/mL with phage buffer. For each assay, 180 uL of adjusted bacterial inocula in TSB were mixed with 20 uL of phage in sterile, untreated Falcon (Corning) 96-well transparent plates to achieve final phage concentrations of 106 PFU/mL, 107 PFU/mL, and 108 PFU/mL. The plates were incubated at 37 C with double orbital shaking in a Tecan Spark 10 M plate reader (Tecan Group Ltd., Männedorf, Switzerland) and growth was monitored by measuring OD550nm at 30-min intervals for 12 h, which results in 25 total time points including the initial (time 0) measurement. Growth curves were obtained by plotting OD after baseline adjustment against time. All assays were performed with three biological replicates. See page 5, paragraphs 1-2. Xie teaches that the host range results produced by the microtiter assay were interpreted and calculated automatically by the plate reader and computer, which reduces potential human errors generated by manual visualization of plaques in the spot assay. See page 13, para 3. Xie further teaches analysis of microtiter plate host range assay data by two equations and presents the results in Figure 1. The results obtained in Xie about the phage host range assay are considered as a phagogram.
Henry teaches development of a high throughput assay for indirectly measuring phage growth using the OmniLogTM system. Henry teaches that the conventional and most accepted plaque assay method of measuring the lytic activity of a phage against its bacterial host is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. The authors describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. The authors show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, they show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies. See Abstract.
Specifically, Henry teaches that automation is one potential solution for the inconsistencies and inconveniences inherent in many of the existing phage assays. The authors tested the utility of the OmniLogTM instrument (Biolog, Inc.) for automated assay of phage growth. The OmniLogTM instrument is a specialized plate incubator (with a capacity of 50 microtiter plates) coupled to a camera and a computer and is marketed for use with specialized phenotypic microarray plates (PM) or specialized plates for bacterial identification assays. In the case of the PM assays, the wells of each plate contain different carbon sources, secondary metabolites and even selective or inhibitory compounds/agents like antibiotics, and redox chemistry is employed to measure respiration of input microorganisms under the various conditions present in each well. The PM technology has been successfully used in a variety of comparative analyses of phenotypes of organisms as well as to assess the accuracy of genome annotations. See page 160, left column, para 2.
Henry teaches that bacterial cultures for the microtiter plate assay were prepared from colonies grown from an overnight culture on Tryptic Soy Agar (TSA) plates and suspended in phosphate buffered saline (pH 7.4) to an optical density of 0.1, which corresponded to roughly 108 colony forming units/ml. After further dilution, 106 CFUs were deposited in each well. Individual phages or a cocktail of Giraffe, BA21, BA28, BA39, BA51 and Gamma were mixed in equal proportions and used to infect the B. anthracis host. The phage titers used ranged from 108 PFUs to 102 PFUs per well, corresponding to a multiplicity of infection (MOI) of 100 to 0.0001. See page 167, left column, para 1.
Henry teaches that Microtiter plates (96 well) plates were prepared as follows. In each well, 90 μl of TS broth mixed with 1% v/v tetrazolium dye was added as growth medium followed by the addition of 10 μl of (109 PFU) phage per well in one column, making a 108 PFU/well dilution. 10-fold dilutions were made in the subsequent columns down to 102 PFU per well, leaving 90 μl left in each well. The last column received media and phage alone, with a total volume of 100 μl. Following dilution of phage, 10 μl of 108 CFU/ml of B. anthracis cells or spores were added to each well designed to have bacteria, increasing the volume to a total of 100 μl and giving a final concentration of 106 CFU per well. The 96 well plates were then incubated in the OmniLogTM system at 37°C for 48 or 72 h. All experiments represent biological replicates of three except the ciprofloxacin experiment which was done once. See page 167, para 2.
Fischer teaches a study on microplate-test for the rapid determination of bacteriophage-susceptibility of Campylobacter Isolates. It teaches that a simple susceptibility test using 800 isolates of one Campylobacter strain with different degrees of susceptibility and four bacteriophages of the British phage typing scheme was developed and examined for its suitability. The test presented is economically cheaper and less time consuming than the conventional agar overlay plate assay and therefore enables the monitoring of changes in the susceptibility pattern during phage therapy under practical field conditions. The main objective of the study was to compare the simplified test with the conventional agar overlay plate assay. The conventional test describes for a population of Campylobacter: i. the rate of resistant isolates (0 plaques) and ii. the degree of susceptibility, also called relative efficiency of plating (EOP), for the remaining isolates. The simplified test divides the isolates into four susceptibility ranks, which are easily distinguishable to the naked eye. Ten Campylobacter isolates out of each rank were subjected to the conventional method for validation of the simplified test. Each resistance rank contained isolates showing certain degrees of susceptibility, reflecting decreasing susceptibility by an increase of the rank. Thus, the simplified test correlated well with the conventional method. See Abstract.
Specifically, Fischer teaches that the susceptibility test was performed in a 668 well microplate (Sigma-Aldrich, CLS3548). Every Campylobacter isolate was tested against all four phages per one individual well and a further well was used for growth control of Campylobacter. Appropriate to routine test dilution (RTD), the phage-containing suspensions were prepared containing 106 PFU/ml of phages 1, 2, 5 or 107 PFU/ml of phage 13, respectively (these concentrations were chosen from preliminary screenings using colonies from the original strain in the Microplate-Test with different phage concentrations, data not shown). The RTD, producing semiconfluent lysis was chosen in order to allow the detection of an increase and decrease of lysis in the Microplate-Test. Afterwards the 100 colonies of the original strain were tested in the Microplate-Test in order to confirm a constant output (Figure 2A). The phage suspensions were stored at 4uC until use. From every phage suspension 10 ml were transferred into the appropriate wells and 10 ml of test Campylobacter suspension, adjusted to McFSt. 5, were added. Subsequently, 0.5 ml of modified NZCYM-overlay composed of 22.0 g NZCYM-powder, 3.5 g Agaragar, 3.5 g Low-melting-agar (Promega Corporation, V2111) per 1000 ml were filled into each well. The plates were incubated under microaerobic conditions after solidification at 4260.5uC for 18 h. See page 3, right column, para 2.
Fischer teaches that the Microplate-Test allows in future partial automatisation for production and reading of a phage susceptibility test for Campylobacter. Compared to the conventional method, the Microplate- Test allows simultaneous examination of a fivefold to tenfold number of isolates, depending on the degree of automatisation. See page 7, left column, para 2.
Accordingly, Xie, Henry and Fischer each teaches a method, as well as a “system”, for measuring sensitivity of a bacterium to a plurality of bacteriophages (i.e., host range of bacteriophages), comprising (1) providing a microwell plate comprising a plurality of wells suitable for containing bacteriophages and host bacteria, (2) dispensing bacterium/phage samples in wells of the microtiter plate allowing incubation of the a mixture of the bacterium and bacteriophage contained in the well for the duration of a first period of time, (3) measuring the assay results by transporting to and introducing at least one mixture in an analytical device and measuring the bacterium-phage interaction of said at least one mixture by said analytical device (e.g., a OD reader of Xie); and (4) interpreting and presenting the measurements by said analytical device in the form of a phagogram by a control unit (the detection devices used in the studies).
However, these prior art references are silent on an analytical device configured to measure the phage-bacterium interaction by qPCR or light emission by luciferin or luciferase.
Paczesny summarizes the recent developments in the field of phage-based methods for bacteria detection (which is equivalent to a bacterium phage sensitivity assay). It focuses on works published after mid-2017. It underlines the need for further advancements, especially related to lowering the detection (below 1 CFU/mL; CFU stands for colony forming units) and shortening the time of analysis (below one hour). From the application point of view, portable, cheap, and fast devices are needed, even at the expense of sensitivity. See Abstract.
Paczesny teaches that conventional microbiological methods for bacteria detection, based on culturing microorganisms, are cheap and selective but also time-consuming and laborious. Therefore, researchers are introducing new detection techniques. Over the past decades various detection methods have been developed including (but not limited to) nucleic acid-based sensors (DNA microarrays, polymerase
chain reaction (PCR) and its derivatives, e.g., multiplex PCR or real-time PCR), immune-based sensors (e.g., enzyme-linked immunosorbent assay [9]) and mass spectrometry sensors. See page 1, para 2.
Paczesny teaches that Nugen group showed detection of E. coli with LOD of around 5 x 102 CFU/mL after two hours incubation with T7 containing NanoLuc luciferase expression cassette. The method required the addition of a substrate for signal generation. Modified phages were prepared by synthetic biology strategy to engineer phages using a simple in vitro method [75]. The method used PCR fragments and in vitro DNA assembly followed by rebooting through transforming into host bacteria and not DNA assembly in yeast. The procedure resulted in the relatively simple and fast preparation of specific phages needed for detecting target bacteria in various applications. See page 7, para 5.
Paczesny teaches that the most often used is a combination of phage amplification and detection of progeny virions employing the PCR technique. Several reports utilizing such an approach were published, with time and limits of detection varying strongly. For instance, Luo et al. [83] showed the detection of Acinetobacter baumannii in serum using p53 phages allowing for LOD in the range of 102 CFU/mL within 4 h. Later they improved the method and achieved LOD of 10 CFU/mL in sputum samples within 6 h [84]. Garrido-Maestu et al. [85] showed the detection of 8 CFU of Salmonella Enteritidis in 25 g of chicken samples within 10 h. Extending the time of the analysis allowed Sergueev and coworkers [86] to achieve LOD of around 1 CFU/mL of Brucella abortus within 72 h in mixed cultures and blood samples. The most inspiring example was published by Anany et al. [87], who developed a phage-based paper dipstick biosensor to detect various foodborne pathogens in food matrices. They used piezoelectric inkjet printing to prepare phage-based bioactive papers that actively lysed their target bacteria. In combination with quantitative real-time PCR, this allowed for a limit of 10 to 50 CFU/mL in the number of various samples with a total assay time of 8 h. See the para bridging pages 8 and 9.
Accordingly, teachings of Paczesny indicate that the sensitivity of a bacterium to a bacteriophage infection may be assayed by methods of qPCR and measuring luciferase light emission, in addition to conventional microbiological methods, such as those disclosed in Xie, Henry or Fischer. However, Paczesny is silent on the use of a multi-well plate.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of Xie, Henry, Fischer, and Paczesny to arrive at the invention as claimed. One would have been motivated to do so, e.g., to detect the phage-bacterium interactions produced on a multi-well plate, as in the studies of Xie, Henry and Fischer, with the qPCR or luciferase light emission test taught in Paczesny, to evaluate the effectiveness of the qPCR or light emission assays as detection method; or, the other way around, one would have been motivated to do so, e.g., to performed the qPCR- or the light-emission-test-based bacterium/phage interaction taught in Paczesny on a multi-well plate, as disclosed in Xie, Henry and Fischer, to evaluate the effect of multi-well plate in the detection of relatively large number of samples (high throughput). Additionally, such a combination, or a substitution of one element for another known in the field to have the same purpose, is evidence that the claimed invention may be found obvious. See MPEP 2144.06. Therefore, the instant invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the references, especially in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL ALLEN, on (571) 270-3497, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/
Primary Examiner, Art Unit 1671