Description:F O R M 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

DEVICE FOR DETECTING ANTIMICROBIAL RESISTANT BACTERIA USING BACTERIOPHAGES
InventorS:

PAL, Sanjay– Citizen of India
MADHAVAN, Ajith– Citizen of India
PORAYATH, Chandni– Citizen of India
BABU, Pradeesh– Citizen of India
SALIM, Amrita– Citizen of India
STANLEY, John*– Citizen of India
THEKKEDATH, Satheesh Babu G* – Citizen of India
NAIR, Bipin– Citizen of US

School of Biotechnology
Amrita Vishwa Vidyapeetham, Kollam - 690019, Kerala,India.

*Amrita School of Engineering,
Amritanagar, Coimbatore – 641112, India
applicants
AMRITA VISHWA VIDYAPEETHAM
School of Biotechnology,
Kollam, Kerala, India 695025

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
 
DEVICE FOR DETECTING ANTIMICROBIAL RESISTANT BACTERIA USING BACTERIOPHAGES
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a divisional application of the complete specification filed on June 13, 2018 and claims priority to Indian provisional patent application no. 201741020669dated June 13, 2017.
FIELD OF THE INVENTION
The present invention relates to amethod, an array device and a kit for the detection of antimicrobial resistant microorganismor multiple drug resistant microorganisms using bacteriophages.
DESCRIPTION OF THE RELATED ART
Antimicrobial resistance (AMR) or multiple drug resistance (MDR) is a growing threat to public health that requires global action. AMR and MDR have mainly advanced due to the evolution of microorganisms, such as bacteria, fungi, viruses, and other parasites, on being exposed to antimicrobial drugs, such as antibiotics, antifungals, antivirals, anti-malarial, and anthelminthic.The misuse and overuse of antimicrobials drugs is accelerating the process of antimicrobial resistance in microorganisms. Other factors that encourage the spread of antimicrobial resistance are poor infection control, inadequate sanitary conditions and inappropriate food-handling. Severalreports implicate wastewater as a 'breeding ground' for antibiotic resistance. Fecal discharge from houses, hospitals and industrial areas into septic tanksand sewage treatment plants constitute metabolized or partially metabolized antibiotics and other drug constituents. Therefore the microorganisms present in the septic tanks and treatment plants are exposed to a wide variety of antibiotics and drugs. This constant exposure of bacteria to antimicrobials functions as a selective environment for the evolution of the microbes into AMR and MDR strains.With increase in the presence of AMR and MDR in wastewater, it has become imperative to develop tools and methods to detect the antibiotic resistant microbes, which can be used for understanding and combating the epidemiology and drivers of AMR.
Bacteriophage amplification has been suggested as a method to accelerate microorganism identification. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replicating themselves. A bacteriophage (or phage) does this by attaching itself to a bacterium and injecting its genetic material into that bacterium, inducing it to replicate the phage from tens to thousands of times. Some bacteriophage, called lytic bacteriophage, rupture the host bacterium releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for infection of a bacterium by parent phage, phage multiplication (amplification) in the bacterium to produce progeny phage, and release of the progeny phage after lysis can take as little as an hour depending on the phage, the bacterium, and the environmental conditions. However, an economically viable and commercially-suitable method for monitoring a plurality of samples for target microorganisms using bacteriophages is currently not available.
The US patent publication US20050250096A1 describes a method of detecting the presence or absence ofantibiotic resistant microorganism in a sample using bacteriophage capable of infecting the target microorganismThe US patent publication US20110183314A1 describes a method of determining the presence or absence of an antibiotic resistant target microorganism in a sample to be tested, by combining with the sample an amount of bacteriophage capable of infecting the microorganism to create a bacteriophage-exposed sample. The time rate of change of the amount of bacteriophage or the change in the rate of change of the amount of bacteriophage serves as an indication of the presence or absence of the microorganism as a function of time.
However, the mode of measurement is based on traditional plaque assays which are both time and labor intensive and often results in poor reproducibility. Also, many bacteriophages do not cause a sufficient level of cellular damage to be visualized using this method. Traditional methods are based on crude assessment methodologies and produces results that vary widely between replicates, obscuring precise quantitative values.
Natecheet al(2006) assesses the performance of the resazurin microtitre assay (REMA) plate method for the detection of antibiotic resistance to isoniazid and rifampicin in 136 clinical isolates of Mycobacterium tuberculosis. Rondónet al(2011) describes a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). The bacteriophage is described as suitable for identifying over 150 strains of Mycobacterium tuberculosis that were resistant to isoniazid, rifampin, and streptomycin.The US patent publication US9624523 discloses a simple culture device that is designed for manufacture and use in areas of limited resources. These devices include culture strips which may be coated or impregnated with antibiotics. These strips may then be used to detect microbes that are resistant to antibiotics. The assay methods employ dormant bacteriophages to qualitatively or quantitatively detect the presence of antibiotic resistant bacteria with resistance to over four antibiotics simultaneously.However, this document does not describe means to identify viability of the antibiotic resistant bacteria. It employs an activatable dormant bacteriophage carrying a reporter gene to qualitatively or quantitatively detect the presence of a substance of interest in a sample. So all bacteriophages targeting different bacteria have to be genetically tagged with the reporter gene thereby, increasing the overall cost and time for development of the method.
Although the above publications employed bacteriophages for detecting microorganisms, they are limited by inability to detect AMR/MDR bacteria specifically, need for expensive phages, reagents and equipment, low sensitivity, low specificity, need for additional steps, development of phage resistance, use of plaque forming assays for detection, and/or lack of automated systems and multiplexing for assaying and detection. Furthermore, DNA amplification by PCR or other methods have technical limitation with respect to multiplexing. Real time qPCR machines can detect only few fluorophores together and thereby only few can be multiplexed. They are also costlier with respect to different tagging chemistry of the fluorophores. Although, few methods in the literature discuss about impedance based detection of phages, the main limitation of the methods are that they do not depend on phage. They rely on the electrical properties of the living bacteria vs. dead bacteria.Additionally, there exists a correlation in different phage groups of resistant and sensitive strain to antibiotic susceptibility. Therefore, the inability of the phages to distinguish between antibiotic resistant and sensitive strains is still a challenge.
SUMMARY OF THE INVENTION
A method, a device and a kit for detecting presence of antimicrobial resistant (AMR) or multiple drug resistant (MDR) bacteriausing bacteriophages are disclosed.
In one embodiment of the disclosure, a method for detecting presence of one or more target antimicrobial resistant (AMR) or multiple drug resistant (MDR) bacteria in a sample using bacteriophages is disclosed. The method comprising treating the sample with one or more antibiotics, the one or more antibiotics are configured to kill or inhibit the growth of antibiotic-sensitive bacteria, combining the antibiotic-treated sample with an amount of a bacteriophage cocktail to create a bacteriophage-exposed sample. The bacteriophage cocktail is derived from a medium containing antibiotic resistant bacteria.The method further comprises maintaining the bacteriophage-exposed sample under conditions supporting amplification of bacteriophages in the one or more target antimicrobial resistant (AMR) or multiple drug resistant (MDR) bacteria and passing the sample through one or more sub-micron filters to obtain a filtered sample. The one or more sub-micron filters are configured to remove bacteria and retain bacteriophages in the sample. Further, adding a volume of the filtered sample to a plurality of wells of an array device, where each well of the plurality of wells is plated with a predefined bacteria and a marker indicative of bacterial viability and incubating the array device under conditions supporting the growth of the bacteria in the wells. The time rate of change of signal from the marker is detected, where a decrease in viability of bacteria is indicative of the presence of the target antimicrobial resistant (AMR) or multiple drug resistant (MDR) bacteria in the sample.
In various embodiments, the target bacteria comprise at least onefrom the group of Escherichia coli, Shigella, Salmonella, Pseudomonas, Klebsiella, Streptococci, or Proteus.In various embodiments, the sample is a body fluid sample from a subject derived from blood, urine, sputum/saliva, or faeces.
In some embodiments, the sample is a waste water sample selected from hospital runoff water, industrial waste water or municipal sewage.
In various embodiments, the sample is pre-treated with antimicrobial agents selected from antibiotics, antifungals, antivirals, antimalarial and anthelmintic.In some embodiments, at least one bacteriophage in the cocktail is tagged with a fluorescent marker.In one embodiment of the invention, a plurality of bacteriophages in cocktail are tagged with distinct fluorescent markers for multiplexing.In one embodiment, the marker indicative of bacterial viability is a resazurin dye.
In various embodiments, the one or more submicron filter comprises a 0.22 micron filter.In some embodiments, the cocktail comprises bacteriophages at a concentration range of 103pfu/mL to 107pfu/mL.In some embodiments, detecting the time rate of change of signal from the marker comprises detecting the signal over a surface area of the well.
In various embodiments, detecting the time rate of change of signal further comprises using an electronic module integrated to the array device through a sensor unit printed at the base of each well, and the sensor unit is configured to detect time rate of change of signal from the marker to determine viability of bacteria.
In one embodiment of the invention an array device is disclosed. The array device comprises a microwell plate comprising a plurality of wells for receiving one or more bacteriophages. Each well comprises a predetermined surface area to be plated with growth media containing target resistant bacteria for each bacteriophage and a marker indicative of bacterial viability. The microwell plate is housed in an environmental chamber supporting growth of the bacteria and a sensor unit printed at the base of each well, the sensor unitdetects and measures the change in impedance over time. The sensor unit is integrated with an electronic meter module to measure and display the concentration of bacteria in the sample at any given time.
In some embodiments, the electronic meter module comprises a microcontroller connected to the each of the sensor unit to convert the output from the sensing unit to the corresponding bacteria concentrations, an output or display unit to display the bacteria concentrations and one or more communication protocols to interface the microcontroller with the output or display units.
In one embodiment, the sensor unit comprises at least one electrode.In some embodiments, the electrodes are carbon based electrodes.In one embodiment of the invention, the device is a microfluidics array device.In one embodiment, the microfluidic array device further comprises optical reader configured for displaying quantitative fluorescence detection.
In one embodiment of the invention, a kit comprising the array and instructions for use thereof to detect antimicrobial resistant (AMR) or multidrug resistant (MDR) bacteria in the sample is disclosed.In some embodiments, the kit comprises bacterial growth media for plating the wells including at least one marker indicative of viability of bacteria.
This and other aspects are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS
The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:
FIG. 1 illustrates a method for detecting antimicrobial resistant (AMR) or multiple drug resistant (MDR) bacteria, according to an embodiment of the present subject matter.
FIG. 2 illustrates an array device with electronic meter module system that measures the bacteriophage concentration in a sample.
FIG. 3shows well plates includingelectrodes at the base of the multiwell plates.
FIG. 4illustrates different stages of the method for detecting AMR bacteria.
FIG. 5shows an equivalent circuit model of the sensing area of the multiwell plates.
FIG. 6 shows phage specificity determination for a) E.coli ET b) E. coli MDR
FIG. 7 shows antibiotic resistance determination by resazurin based dye.
FIG. 8A shows phage array with known host, Salmonella against a specific phage.
FIG. 8B and 8C show bacteriophage effect against Salmonella in bacterial cocktail determined using resazurin with different dilutions of the host target, Salmonella.
FIG. 9A-9C show Salmonella-specific phage effect when treated withbacterial cocktail of 9A: Vibrio,9B:Shigella,and 9C: Salmonella.
FIG. 9D-9Eshow bacteriophage effect against specific targets in sewage determined using resazurin for 9D: E. coli MDR and 9E:Klebsiella.
FIG. 10A-10E show the phage cocktail effect against specific targets in heterogeneous bacterial cocktail determined using resazurin10A: E. coli ET,10B: Salmonella,10C: E. coli MDR,10D:Klebsiella,and10E:Shigella.
Referring to the drawings, like numbers indicate like parts throughout the views. 
DETAILED DESCRIPTION
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.
Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.The word “exemplary” is used herein to mean “serving as an example, instance, or illustration”. Any implementation described herein as “exemplary” is not necessarily to be construed as advantageous over other implementations.
The present inventionin its various embodiments discloses a method, a device and a kit for detecting the presence of antimicrobial resistant (AMR) microorganisms or multiple drug resistant (MDR) microorganisms using bacteriophages.Bacteriophages may be isolated, tested and used for the detection of a particularhost strain selectively. Bacteriophage or phage based sensors are advantageousformonitoring a heterogeneous environment like waste water as theyare economically viable. Phages are self-replicating and highly specific in their host interaction and hence ensure suitable signal amplification required for an ideal sensing element.
Amethod of detecting one or more target antimicrobial resistant (AMR)or multiple drug resistant (MDR) bacteria in a sample100using bacteriophages is illustrated inFIG. 1, according to one embodiment of the invention.The method 100includes, treating the sample with one or more antibiotics in block 101, wherein the one or more antibiotics are configured to kill or inhibit the growth of antibiotic-sensitive bacteria. The antibiotic treated sample is mixed with an amount of cocktail of bacteriophagesin block 102 to form a bacteriophage exposed sample. The bacteriophage exposed sample is maintained under conditionsthat support amplification of bacteriophages in one or more target bacteria in block 103. The sample is then filtered using sub-micron filters in block104 to separate thebacteria and bacteriophages. A specific volume of the filtered sample containing the phages is added to a multi well plate array including a plurality of array elements along with a marker or dyes indicative of bacterial viabilityin block 105. In one embodiment, the marker is a reagent for quantifying the viability of the bacteria, such as redox fluorescent dye, typically a resazurin dye. The amplified phage sample in the cocktail is recognized by monitoring the type of AMR/MDRbacteriain the multi well plate.
The mixture is then incubated in the array device under conditions that support the growth of the bacteriain block 106. The time rate of change of signal from the marker is detected, in block 107 and the strains of the bacteria are identified by correlating the reduction in the viability in the test well with that of a control well containing phages of predetermined titre. The detection of time rate of change of signal from the marker includesdetecting the signal over a surface area of the well. The fluorescence obtained is directly proportional to the predetermined bacterial population in the well. The reduction in the fluorescence may be due to bacterial infection and lysiswhich is correlated with the titer of phages. The reduction in fluorescence isindicative of presence of AMR or MDR bacteria in the sample corresponding to the identified strain from the multi well plate.
In some embodiments the plurality of bacteriophages in cocktail are each additionally tagged with distinct fluorescent markers for additionally detecting phage amplification in the filtered sample.
In some embodiments, the signal is detected by an electrode sensor attached to the microwell plate and impedance is recorded by a connected electronic meter module. In some embodiments, the method is semi-automated or completely automated using a microfluidics module.
In one embodiment, the sample may be a sample obtained from a subject such as a body fluid sample. In other embodiments, the sample is obtained from a water source such as sewage sample. In some embodiments, the sample is any water or waste water in which antimicrobial resistance is to be identified, such as body fluid sample, a fluid sample from a location such as a septic tank, a sewage treatment plant, a hospital sewage line or other wastewater location. In one embodiment, the sample containing one or more AMR/MDR microorganisms is obtained by treating biological matter with antimicrobial agents, such as antibiotics, antifungals, antivirals, antimalarial, or anthelmintic.
In one embodiment, the sample is treated with a suitable amount of one or more antibiotics including, but not limited to, ampicillin, tetracycline, chloramphenicol, or penicillin prior to bacteriophage cocktail treatment. In one embodiment, the antibiotic-treatment results in selective killing of non-target bacteria which are sensitive to the antibiotic thereby enhancing the specificity in detecting the antibiotic resistant bacteria. For instance, if AMR/MDR bacteria of a particular strain of E.colistrain are to be detected and monitored, then a bacteriophage that specifically targets AMR/MDRE.colistrain is selected and added to the cocktail. In one embodiment, a phage stock library is prepared and characterized upstream for identifying the specific strains. The phage library may be prepared by isolating and selecting phages from a natural environment or source. Accordingly, the cocktail is custom configured to contain bacteriophages tailored to target a specific strain of AMR/MDR bacteria. In various embodiments, the bacteriophage cocktail is derived from a medium containing antibiotic resistant bacteria. In some embodiments, the medium containing antibiotic resistant bacteriais derived from bodily fluid or discharge such as blood, urine, sputum/saliva, or faeces. In some embodiments, the medium containing the antibiotic-resistant bacteria is derived fromhospital runoff water, industrial waste water or municipal sewage.
In various embodiments the target bacteria includesat least one selected from the group of E.coli, Salmonella, Klebsiella, Shigella, Pseudomonas, Vibrio, Streptococci, or Proteus etc. In various embodiments, the sample is pre-treated with antimicrobial agents selected from antibiotics, antifungals, antivirals, antimalarial and anthelmintic.
An array device 200 for detecting antimicrobial resistant (AMR) or multiple drug resistant (MDR)microorganisms using phages is illustrated in FIG.2, according to one embodiment of the invention. The array deviceis connected to an electronic meter module system that measures the bacteriophage concentration in a sample.The array device 200 includesmultiwell plates201such as microtiter plates includinga plurality of cell culture wells for receiving one or more bacteriophages. Each well 201 includesa predetermined surface area301 configured to be plated with growth media containing a target strain of bacteria for each bacteriophage. The multiwellplates are housed in an environmental chamber 302 supporting bacterial growth. A sensor unit 202 is printed at the base of each welland the sensing unit includes a plurality of electrodes with at least one active electrode 303, one reference electrode 304andat least one counter electrode 305.When the target bacteria are infected by bacteriophages, lysis of said bacteria takes place and a change in impedance value of the electrode occurs based on the number of bacteria lysed. This change in impedance between the working electrodeand the reference electrode over time is detected and measured which is anindicative of bacterial count in the biological sample. The change in impedance value is dependent onthe bacterial lysis which in turn is proportional to the concentration of the bacteriophages in the sample. FIG. 3 shows well plates includingelectrodes at the base of the multiwellplate.In some embodiments, the electrodes are preferably, carbon based electrodes. The device further includesan electronic meter module 203 integrated with the sensor unit 202 to measure and display the antimicrobial resistant microorganism concentration in the fluid sample.
In one embodiment, the electronic meter module 203includesa microcontroller 204 connected to each of the sensor unit 202 to convert the output from the sensing unit to the corresponding bacteria concentrations. The meter module 203 further includesan output or display unit 205that includes Bluetooth module, graphical LCD etc. to display the bacteria concentrations and one or more communication protocols 206 to interface the microcontroller with the display or output units. In some embodiments, the module furtherincludesone or more programmable analog front end (AFE) 207 ICs connected to the microcontroller. The AFEs 207 are further connected to the sensor unit202located in each of the individual wells of the multiwell plate and receive the impedance change valuemeasured in one or more samples from one or more well plates. The microcontroller204 is connected to the AFE207 and the change in impedance from the AFE’s is fed to separate ADC channels of the microcontroller. The microcontroller204 also controls the communication protocol module206 that includes SPI, I2C and USART etc.The microcontroller is programmed to convert the change in impedance into the corresponding bacteriophage concentrations. In some embodiments, the number of AFEs in the system is equal to the number of wells that contain the sample. In one embodiment, the module includes at least 96 wells and 96 AFEs to measure the bacteriophage concentration in the sample.
In one embodiment, the device is a microfluidics array device, wherein the device includesmultiwellplates, microfluidic channels for fluid communication with a plurality of inlet ports and outlet ports, at least one pressure source configured to cause a predetermined amount of fluid to flow from the inlet ports to the microfluidic channel and a controller to control the pressure and amount of fluid flowing through the channel. A pressure differential is generated by the pressure sources, such as negative pressure source, for example vacuum. The antibiotic treated sample and bacteriophage cocktail are drawn by negative pressure through specific ports in the channel and mixed thoroughly. A marker indicative of bacterial viability is added to the mixture through a port. The difference in the fluorescence or impedance is detected and the microorganism concentration is displayed in the output or display unit.The device further includesan optical reader configured for displaying quantitative fluorescence detection. The microfluidic array device further includesstorage units for the phage stock powder, bacterial stock powder, bacterial media powder, sterile water, syringe, filter of 0.22 micron, marker or dye such as resazurin powder and one or more antibiotics.
In some embodiments, amethod of detection of antimicrobial resistant (AMR) or multi drug resistant (MDR) enteric microorganismssuch as bacteria in sewage with a cocktail of bacteriophages 400 is disclosed. The method includes treating a fluid sample with one or more antibiotics401, wherein the one or more antibiotics are configured to kill or inhibit the growth of antibiotic-sensitive microorganisms,and providing the antibiotic-treated sample 401 with a cocktail of bacteriophages402 uniformly within the elements of a control array 403and a test array 406. In some embodiments, multiple fluid samples from various tagged sourcesare tested. The cocktail of bacteriophages402 are used as the sensing element to screen MDR strains of microorganisms. The cocktail of bacteriophages 402includes various strains of AMR or MDR bacteria 402-1, 402-2, 402-3 … 402-n,where402-1 is a known bacteriophage cocktail with a specific titre that represents a basal level against which the results are to be interpreted. FIG. 4 illustrates a schematic diagram of phage based detection of multiple drug resistant (MDR) microorganisms based on their lysis and the subsequent comparison of fluorescence with the control array.
In various embodiments,the control phage array 403is seeded with the antibiotic-treated sample401 and cocktail of bacteriophages 402 to lyse various strains of AMR or MDR bacteria 402-1, 402-2, 402-3 … 402-n,wherein 402-1 is the known phage cocktail.The bacteriophages attach to the bacterium in the sample and differential amplification of the bacteriophages 404 takes place. The control phage array 403is an array of target bacteria and detects the difference in the titre. The reduction in the fluorescence due to bacterial respiration is correlated with the titre of phages which represents the basal level.
Subsequently, the antibiotic-treated sample401 with cocktail of bacteriophages 402 iscombinedto create a bacteriophage exposed sample that manifests the activity of the phages against the specific strains of drug resistant microorganisms to be identified in the samples. The bacteriophage exposed sample is maintained under conditions that support amplification of bacteriophages in one or more target bacteria. The bacteriophages attach to the bacterium present in the sample and insert its genetic material into the bacterium cell to destroy or lyse the cell, releasing new phage particles 404.Consequently, differential enrichment of the bacteriophages takes place based on the presence of specific groups of bacteria and the signature titre changes. Then it is passed through a 0.22micron bacterial filter 405 to remove the bacterial population in the sample. The preparation obtained represents a lysate of differentially amplified bacteriophages based on the type of MDR strains present in the sewage.
In various embodiments, a sensitive redox dye like resazurin based assay is used to detect the growth, inhibition, lysis, multiplication and/or metabolic activity of the bacteria and the changes in comparison to the control that helps in establishing the type of MDR strains present in the test samples. Titre detection of the lysate using redox fluorescent dye reveals the changes in comparison to the control 403 and helps in establishing type of MDR strains present in the test samples 406. The specific titre difference is detected to establish the identity of AMR or MDR microorganisms. The information about the types of resistant microorganisms is then transduced to differentially amplified titre, as compared to the basal level titre.
In some embodiments, the activity is monitored over the period of time to identify the presence of AMR microorganisms. The activity indicating the presence of the MDR or AMR is elicited using various types of tagging. The viability of the microorganisms is monitored for a predetermined time across the array 406 by comparing with a control array 403 that includes phages of predetermined titre. The color intensity 407 is proportional to the viability of the microorganisms. The fluorescence obtained is inversely proportional to the titre of the bacteriophages indicating reduction in metabolic activity of the bacteria with reference to the control.The reduction in viability indicates the presence of respective MDRs and the specific strain is identified.
In various embodiments, fluorescent tagging of the phages in cocktail ensures high sensitivity and is additionally amenable for multiplexing.In one embodiment, the sensing unit monitors with high specificity, sensitivity, and capability for multiplexing in a heterogeneous environment like wastewater.
In one embodiment of the invention, the specific bacteriophages that target only the drug resistant bacteria are isolated leaving out the sensitive ones. The AMR specific bacteriophages that are enriched in sewage or body fluid are filtered. The phage filtrate is then titrated in defined host library in multiwell.
In one embodiment of the invention, the antimicrobial resistance exhibited by bacteriophages in the sensor array is determined by electrochemical impedance method. In various embodiments, the sensor unit includesat least one active electrode, reference electrode and counter electrode and the electrodes are configured to measure the electrochemical impedance over the well area. In embodiments of the method, an AC current with a frequency sweep is applied across one or more cells of the array to determine the changing impedance value of the media. The impedance value changes according to the concentration of the suspension medium at the sensing area. The concentration of the suspension medium changes according to the number of bacteria present in the medium of each sample. FIG. 5 shows an equivalent circuit model of the sensing area of the microwellplates. The sensing area of the individual plate 500 is modeled electrically as cell impedance Zcell501 in parallel with the impedance contributed by all materials between the electrodes 503, that consist of a solution resistor Rm in parallel with the capacitance of double layer Cdl. The impedance Zcell501 and impedance contributed by all materials 503 between the electrodes are in series with a pair of electrode resistances Re505. The Zcell501 represents a cytoplasm resistor (Rc) in series with a membrane capacitor (Cc). The overall impedance of the measurement system ZT is given by,
Z_T=2R_e+1/(1/R_m +jωC_dl+1/(R_c-jωC_c ))
whereω is the angular frequency of the electrical signal. ZT changes according to the number of bacteria present in the medium of each sample.
In various embodiments, a kit includingthe array device and instructions thereof to detect antimicrobial (AMR) or multiple drug resistant (MDR) bacteria in the sample are disclosed. The kit includesbacterial growth media for plating the wells including at least one marker indicative of viability of bacteria.
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. Further, the examples presented here are illustrative of the embodiments and are not to be construed as limiting the scope of the invention as delineated in the claims to follow.
EXAMPLES
Example 1: Materials and methods
Growth medium for bacterial cultures and Bovine Serum Albumin (BSA) were purchased from Himedia Laboratories. Resazurin and other reagents were procured from Sigma Aldrich. E. coli, ET strain, a clinical isolate, gifted by Dr. Bhabatosh Das, THSTI, Faridabad was used for the selection and propagation of bacteriophages. Clinical strains of Shigelladysenteriae, Salmonellaenterica, Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae and a multidrug resistantE. coli,(E. coli MDR obtained fromDr. Anil Kumar V, Microbiology department, AIMS, Kochi, Kerala)were usedto check the cross infectivity (tropism) of the isolated bacteriophages.. Bacterial strains were maintained in Luria-Bertani(LB) agar plates at 37º C. In liquid cultures bacterial strains weregrown in LB broth with 120 rpm shaking.
Example 2: Isolation and characterizationof bacteriophages
A sample of settled sewage water was filtered using 0.22µm pore size syringe filter and filtrate was enriched by adding 2.5 ml of Deca strength phage broth into 22.5 ml of filtrate and inoculating 1 ml of overnight grown host culture (E. coli ET). The resulting preparation was incubated at 37º C for 24 hours, after which the culture was centrifuged to pellet down the bacteria and the supernatant was filter sterilized using 0.22µm syringe filter. The supernatant was checked for the activity on the E. coli culture by spot assay where 10µl of the supernatant was spotted onto the surface of the host bacterial lawn, left to dry and incubated overnightat 37º C. After incubation, the plates were checked for zone of lysisindicating the presence of phages.
Example 3: Estimation of titre of the bacteriophages
The titer of the bacteriophages against E. coli ET from enrichedsewage was determined by plaque assay using agar overlay method. The bacteriophages with titer of 103pfu/mL to 107pfu/mL were used. The filter sterilized phage lysates were diluted in SM buffer(100 mM NaCl, 8 mM MgSO4•7H2O, 50 mM Tris-Cl (pH 7.5) and0.01% (w/v) gelatin solution) to 10−8dilution and 100µl each ofthe diluted phages and the host bacteria (overnight culture) weremixed with 4 ml of soft agar (0.7% agar) which were then over-laid onto the solidified base LB agar plates (2% agar) and incubatedovernight at 37º C.Individual plaques formed after incubation were further pickedand enriched separately against E. coli ET and their respective titerswere determined.
Example 4: Phage specificity determination
The specific action of the phage in different E. coli strains was done using two different E. coli strain, one of which is the MDR strain and the other one the clinical isolate of the E. coli ET. FIG. 6 shows the phage specificity for E. coli ET and E. coli MDR. The phage has been isolated against the E. coli ET strain which is not cross reactive against the other MDR host. The E. coli ET phage was treated with both the clinical and the MDR strains and the fluorescence was observed at regular interval of time for 1 hour. The results showed that the phage was acting very specifically against its specific host and not to the other strain.
Example 5: Antibiotic resistance determination by resazurin based dye
Antibiotic resistance profiling is done for the pure culture based on the increase in the fluorescence for the resazurin dye. E. coli ET which is sensitive to kanamycin and zeocin was used and water as control change in fluorescence has been measured. FIG. 7 shows the fluorescence measurement of control, kanamycin and zeocin over a period of time of 2 hours. It was found that the E.coli ET strain is sensitive to both kanamycin and zeocin,
Example 6: Phage effect in synthetic mixture:
In order to mimic the sewage micro biome, a synthetic mixture of microorganism consisting of Salmonella, Pseudomonas and E.coli ET and the phage against the Salmonella, been treated with the synthetic mixture was constituted. FIG. 8A shows the phage array with known host, Salmonella against the specific phage. The bacteriophages with titer of 106pfu/ml for Salmonella were used. The synthetic mixture on the basis of different dilutions of the host target, Salmonella is reconstituted and treated with same titre of phages and the resultant filtrate was treated with a host array.FIG. 8B and 8C shows bacteriophage effect against Salmonella in bacterial cocktail determined using resazurin with different dilutions of the host target, Salmonella. The results showed a decrease in the viability of the host as the target organism increases in the test was noted.
Example 7: Phage effect on different dilution of targets:
Salmonella specific phage was treated with the synthetic mixture of the bacterial cocktail including Salmonella, Vibrio and Shigella with varying concentration of the host Salmonella and incubated for 45 minutes. The phages amplified were isolated from the mixture for testing phage sensitive assay. FIG. 9 shows Salmonella specific phage effect when treated with bacterial cocktail of Vibrio, Shigella and Salmonella. The results showed that there is a correlation with the phage targets and the array fluorescence with the target host Salmonella but there is no correlation with the non-target hosts Vibrio and Shigella.
Example 8: Bacteriophage effect against specific targets in sewage determined using resazurin:
The sewage have been augmented with E. coli MDR, resistant to Ampicillin, Tetracycline and chloramphenicol and the augmented sewage was treated with these antibiotics at high concentration and incubated for a period of 1 hour followed by the MDR phage treatment for another 45 minutes and the lysate were titrated against the MDR and Klebsiella in the array. FIG. 9B shows the bacteriophage effect determined using resazurin. The results shows that a reduction in the MDR viability is seen when compared to the control and there is no reduction in the test and control in the Klebsiella control.
Example 9: Phage cocktail effect against specific targets in a synthetic heterogeneous bacterial cocktail:
The phage cocktail was treated with the heterogeneous synthetic mixture of E. coli ET, Salmonella, E. coli MDR, Klebsiella and Shigella and the filtrate of the reaction was analyzed for the phage titre by examining the difference in the test and the control signal produced by the resazurin in the phage array platform for the individual target organism. FIG. 10 shows the phage cocktail effect against specific targets in heterogeneous bacterial cocktail determined using resazurin for E. coli ET, Salmonella. E. coli MDR, Klebsiella and Shigella.
, Claims:WE CLAIM:

13. An array device (200), comprising:
a microwell plate (201) comprising a plurality of wells for receiving one or more bacteriophages, wherein each well comprises a predetermined surface area (301) configured to be plated with growth media containing a target resistant bacteria for each bacteriophage and a marker indicative of bacterial viability, and wherein the microwell plate is configured to be housed in an environmental chamber supporting growth of the bacteria; and
a sensor unit (202) printed at the base of each well, wherein the sensor unit is configured to detect and measure the change in impedance over time;
wherein the sensor unit is configured to be integrated with an electronic meter module (203) integrated with the sensor unit (202) and configured to measure and display the concentration of bacteria in the sample at any given time.

14. The device of claim 13, wherein the electronic meter module (203) comprises:
a) a microcontroller (204) connected to the each of the sensor unit (202) and configured to convert the output from the sensing unit to the corresponding bacteria concentrations;
b) an output or display unit (205), configured to display the bacteria concentrations; and
c) one or more communication protocols (206) configured to interface the microcontroller with the output or display units.

15. The device of claim 13, wherein the sensor unit (202) comprises at least one electrode.
16. The device of claim 13, wherein the electrodes are configured to measure the electrochemical impedance over the well area.

17. The device of claim 13, wherein the electrodes are carbon based electrodes.

18. The device of claim 13, wherein the device is a microfluidics array device.

19. The device of claim 18, wherein the microfluidic array device further comprises optical reader configured for displaying quantitative fluorescence detection.

20. A kit comprising the array device of any one of claims 13-19, and instructions for use thereof to detect antimicrobial resistant (AMR) or multidrug resistant (MDR) bacteria in the sample.

21. The kit of claim 20, further comprising bacterial growth media for plating the wells including at least one marker indicative of viability of bacteria.

Dr V. Shankar IN/PA-1733
For and on behalf of the Applicants