WO2012158041A1 - Diagnostic methods and kits for determining the presence of a microorganism in a sample - Google Patents
Diagnostic methods and kits for determining the presence of a microorganism in a sample Download PDFInfo
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- WO2012158041A1 WO2012158041A1 PCT/NL2012/050352 NL2012050352W WO2012158041A1 WO 2012158041 A1 WO2012158041 A1 WO 2012158041A1 NL 2012050352 W NL2012050352 W NL 2012050352W WO 2012158041 A1 WO2012158041 A1 WO 2012158041A1
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- microorganism
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- phage
- salmonella
- lysed cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
Definitions
- the invention relates to methods and kits for detecting a microorganism in a sample.
- Agents such as phage, capable of lysing the microorganism are employed.
- Contamination by microorganisms is a major cause of food and water-borne infections globally causing gastroenteritis, diarrhea, cramps, vomiting and often fever, such as that referred to as "food poisoning" to "life -threatening disease” .
- Illness in humans often results from the eating of undercooked meats, milk or eggs or from cross -contamination of other foods which are eaten without cooking.
- the most commonly recognized food borne infections are those caused by bacteria such as, Salmonella, Listeria, Campylobacter, Staphylococcus aureus and E. coli 0157:H7.
- Salmonella is found throughout the environment, particularly in the intestinal tracts of birds, reptiles, and farm animals.
- the principle bacterial pathogens that have been shown to cause human intestinal disease associated with food poisoning include Bacillus cereus, Salmonella enterica spp, Listeria monocytogenes, Vibrio paraheamolyticus, enterotoxigenic £ . coli, Campylobacter spp., Staphylococcus aureus, Yersinia enterocolitica, and Clostridium perfringens.
- Conventional methods for the detection of contamination employ non-selective or selective culturing, or enrichment, followed by plating the cultures on selective media for verification of suspect colonies. This approach is time consuming and can take several days before results are obtained. Improved diagnostic methods and kits are therefore needed for the detection of microorganism in a sample, such as a bacterial contaminant in food, water, environmental, medical, agricultural, veterinary, pharmaceutical or industrial fermentation preparations.
- the present disclosure provides rapid and highly sensitive methods and kits for the detection of microorganisms in a sample.
- the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to an agent capable of lysing said target microorganism; providing conditions that permit the agent to lyse the microorganism, if present; and detecting the presence of non-lysed cells in the first and second parts.
- said agent is a phage and the method comprises the steps of exposing the first part of the sample to a phage capable of infecting said target microorganism and providing conditions that permit the phage to infect and lyse the microorganism.
- the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism, if present;
- the microorganism is a bacterium and the phage is a
- a decrease in cells in the first part in comparison to the second part indicates the presence of the microorganism in the sample.
- the methods further comprise exposing the sample to a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism such that the microorganism becomes associated with the solid substrate.
- the solid support is
- the binding agent is an antibody.
- the methods further comprise one or more growth steps.
- one or more of the growths steps is a selective growth step.
- the methods further comprise at least one pre-enrichment growth step and/or at least one selective growth step, preferably the selective growth step follows the pre-enrichment step.
- the methods further comprise exposing both first and second parts to a reagent that labels non-lysed cells and detecting said reagent to determine the presence of non-lysed cells in the first and second parts.
- the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labelled binding agent, such as an antibody, capable of binding the microorganism.
- the reagent that labels non-lysed cells is detected using flow cytometry.
- the target microorganism is Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22- like, T7-like, epsilonl5, KS7, Felix 01 bacteriophage, or a combination thereof.
- the phage is Felix 01.
- the disclosure provides diagnostic kits for the detection of a target microorganism in a sample comprising an agent that specifically lyses a target microorganism, preferably a phage, and a reagent that labels non-lysed cells.
- the kit further comprises a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism if present in the sample such that the microorganism becomes associated with the solid substrate.
- the solid support is immunomagnetic beads.
- the binding agent is an antibody.
- the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labeled binding agent, such as an antibody, capable of binding the microorganism.
- the kit further comprises a microorganism susceptible to lysis by the lysing agent, e.g. a microorganism susceptible to infection by said phage.
- the kit further comprises further a microorganism that is not susceptible to infection by said phage.
- the microorganism is a bacterium and the phage is a bacteriophage.
- the target microorganism detected by the kit is
- Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22-like, T7-like, epsilonl5, KS7, Felix 01 virus, or a combination thereof.
- the microorganism is a bacterium and the phage is a
- Phages are viruses that have evolved in nature to use microorganism as a means of replicating themselves.
- a bacteriophage for example, 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 bacteria releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for phage infection of bacteria, phage multiplication
- U. S. Patent 5,888, 725 describes a method utilizing unmodified, highly specific lytic phages to infect target bacteria in a sample. Phage-induced lysis releases certain nucleotides from the bacterial cell such as ATP that can be detected using known techniques.
- U.S. Patent 6,436,661 describes a method whereby a phage is used to infect and lyse a target bacterium in a sample releasing intracellular enzymes, which react in turn with an immobilized enzyme substrate, thereby producing a detectable signal.
- Alternative methods detect the bacteriophage amplified in a target microorganism.
- WO 2006/083292 describes the detection of bacteriophage protein or nucleic acid. While these methods have the advantage of using unmodified phage, the sensitivity of the assays is limited as the concentration of detected markers (nucleotides or enzymes) is directly proportional to the concentration of target bacteria in the sample. These methods also often have a relatively high background level due to the presence of the detected components in the sample. For example, meat also contains enzymes and nucleotides.
- the present disclosure provides methods and kits for detecting
- the disclosure provides methods for detecting the presence or absence of a microorganism in a sample comprising dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present; and detecting the presence of non-lysed cells in the first and second parts.
- any agent capable of specifically lysing a target microorganism may be used in the methods and kits disclosed herein.
- Preferred agents are the phages disclosed herein.
- microorganism include, e.g., antibiotics and bactericides.
- said agents may be coupled to, e.g., antibodies that specifically recognize the target microorganism.
- the methods described herein comprise additional steps that may increase the sensitivity of the assay.
- methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present;
- the agent preferably a phage will lyse the microorganism if present, thereby decreasing the number of intact cells in the first part of the sample in comparison to the second part of the sample.
- the presence of a phage will lyse the microorganism if present, thereby decreasing the number of intact cells in the first part of the sample in comparison to the second part of the sample.
- microorganism in a sample is indicated by a greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, preferably greater than 85%, or greater than 90% reduction in cell number in the first part of the sample, i.e., agent treated, as compared to the untreated part of the sample.
- microorganisms refer to bacteria, mycoplasmas, and other microscopic living organisms.
- the microorganisms are bacteria.
- Target bacteria contemplated by the present methods and kits include, but are not limited to, bacterial cells that are food, water, or environmental
- detection of bacteria relates to a single species, isolate, serovar or strain as well as groups or families of organisms.
- Bacteria include, but are not limited to, Acinetobacter spp., Actinomyces spp., Bacillus spp. (e.g., Bacillus anthracis, Bacillus subtilis, Bacillus cereus), Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucellae (e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella canis),
- Acinetobacter spp. e.g., Actinomyces spp.
- Bacillus spp. e.g., Bacillus anthracis, Bacillus subtilis, Bacillus cereus
- Bordetella pertussis e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella canis
- Brucellae e.g., Brucella melitensis, Brucella ab
- Costridium spp. e.g., Clostridium difficile, Clostridium perfringens
- Staphylococcus aureus Staphylococcus epidermidis
- Shigella spp. e.g., Shigella dysenteriae
- Spirillum minus Streptococci
- Treponema pallidum Ureaplasma urealyticum
- Vibrio spp. e.g., Vibrio cholerae, Vibrio vulnificus
- Xanthomonas maltophilia e.g., Yersinia pestis.
- bacteria are selected from Salmonella; E. coli, preferably E. coli 0157:H7; Listeria, preferably L. monocytogenes; Legionella and Campylobacter.
- the genus Salmonella comprises two species, Salmonella bongori and
- Salmonella enterica Salmonella enterica is divided into serovars or serotypes, based on somatic O-antigens, flagellar (H) antigens, and surface antigens. There are at least 1500 food-borne Salmonella serovars identified so far.
- Salmonella serotypes detectable using the methods and kits described herein include, for example, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Java, Salmonella enterica serovar Infantis, Salmonella enterica serovar Virchow, Salmonella enterica serovar Mbandaka. Salmonella enterica serovar Livingstone,
- Salmonella enterica serovar Senftenberg Salmonella enterica serovar Hadar, Salmonella enterica serovar London, Salmonella enterica serovar Rissen, Salmonella enterica serovar Anatum, Salmonella enterica serovar Bredeney, and Salmonella enterica serovar Derby. While all Salmonella enterica serotypes are considered potentially pathogenic to humans, only a few are pathogenic to animals. They are often the inhabitants of healthy poultry and pigs, which serve as reservoirs for human transmission. Some serotypes do demonstrate host specificity. The identification of a particular subspecies or serotype can therefore provide information regarding, for example, the contamination route.
- the disclosure provides methods as described herein for detecting the presence or absence of one or more bacterial serotypes in a sample by using one or more bacteriophages that are capable of distinguishing between one or more serotypes.
- serotype and serovar refers to bacteria within the same species that can be distinguished on the basis of their surface antigenic properties.
- Samples include, e.g., various food products, water, industrial or agricultural products, biological samples such as urine, feces, sputum, blood or tissue samples, and may be solid or liquid samples.
- Phages preferably lytic phages, are employed in the methods and kits described herein due to their high degree of specificity when infecting a microorganism.
- Lytic phages also includes phages which are normally non- lytic but have been modified to induce lysis in the host microorganism. A method for the modification of such phages is described in U.S. Patent
- a phage capable of infecting said target microorganism refers to the ability of the phage to infect and replicate within the host and ultimately destroy the cell while releasing numerous phage progeny into the medium.
- the term phage includes, for example, bacteriophages,
- mycobacteriophage and mycoplasma phage.
- the phage is a bacteriophage.
- the specificity of a phage for its host is determined at two levels.
- the first level of control involves the interaction of phage components with
- a phage is specific for a given host when it is capable of infecting the given host and does not infect cells of another species or strain.
- microorganism refers to conditions associated with, for example, time, temperature, and suitable buffering conditions that are well-known to one skilled in the art. (See for example Sands JA, et al., 1974 Biochimica et
- Example 1 of the present disclosure The methods and kits provided herein can also be used to determine the presence or absence of more than one microorganism or more than one bacterial serotype in a single assay by using one or more phages.
- a single phage can recognize, for example, more than one bacterial serotype.
- a combination of phages is used that recognize different host species, different host strains, or preferably different host serotypes.
- Phage useful in the methods and kits described herein also may include genetically modified or recombinant phage that have been altered to increase binding affinity, infectivity, burst size, multiplicity of infection or lytic ability for the microorganism to be detected.
- the phages useful in the methods and kits described herein have not been genetically modified to express a report molecule.
- the phage is not genetically modified.
- a wide variety of bacteriophages are available for any given bacterial cell, for example, from the American Type Culture Collection (ATCC, P.O. Box 1549 Manassas, Va., USA) or by isolation from natural sources that harbor the host cells. A list of phage types is published as the Catalogue of Bacteria &
- Bacteriophages (American Type Culture Collection, Rockville, Md. 1989). Over 20,000 strains of bacteria (with 7,000 being Salmonella) have been evaluated for phage identification (He and Pan, 1992, J Clin Microbiol 30(3): 590-4).
- the phage is Felix 01.
- Additional Salmonella bacteriophages and method for isolating phages are described in PCT Publication WO2005/024005 and Capparelli R et al., J Infect Dis. 2010 Jan 1;201(1):52-61 A skilled person is aware of additional Salmonella bacteriophages and method for isolating phages.
- Salmonella phages which are described, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
- Additional bacteriophages include, but are not limited to
- Actinoplanes/Micromonospora phages (Ap3, Ap4, Mml, Mm3, Mm4, Mm5, phiUW 51); Amycolatopsis phages (W2, W4, W7, Wll); Bacillus phages (GA-1, Phi 29, SP.beta.); Campylobacter phages (e.g., NTCC 12669 , NTCC 12670 , NTCC12671 , NTCC12672 , NTCC12673 , NTCC12674 , NTCC12675 ,
- NTCC12676 NTCC12677 , NTCC12678 , NTCC12679 , NTCC12680 ,
- Saccharothrix phage (Wl); Sporichthya phage (Spl); Streptomyces phages (P23, P26, SI, S2, S3, S4, S6, S7, SH10, phi A. streptomycini III, phi8238, phiC31); Terrabacter phages (Tbl, Tb2); Tsukamurella phage (Tsl).
- Other suitable phages are known to one of skill in the art and may be found, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
- Phages specific for particular bacteria can also be selected using routine techniques in the laboratory due to the ability of the phage to rapidly mutate, thereby producing host range mutants.
- lysed cell and “non-lysed cell” are well-understood to one of skill in the art.
- a non-lysed cell has a cell wall significantly intact to allow its detection by various assays known to one of skill in the art.
- these devices rely on the use of a single sensor (e.g., pH or carbon dioxide indicator) in a layer adjacent to a layer of growth medium for detecting the presence of a bacterium.
- a single sensor e.g., pH or carbon dioxide indicator
- the presence of non-lysed cells is detected using a reagent that labels non-lysed cells.
- Suitable reagents include reagents which are permeable to cells, e.g., SYBR Green, oxazole yellow, thiazole orange, ethidium bromide, fluorescein diacetate,and PicoGreen.
- reagents for labelling non-lysed cells include labelled-binding agents capable of binding the microorganism.
- the binding agent may preferentially bind the target microorganism over non- target microorganisms or may bind microorganisms indiscriminately.
- Binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody,
- antibodies include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies, artificial antibodies, and phage display-derived antibodies.
- binding agents against various microorganisms, as well as methods of generating binding agents are well known in the art. The binding agents may recognize, for example,
- Suitable binding agents include, e.g., anti-lipid A antibodies (AbCam # ab20001). In some embodiments, the binding agents recognize intracellular components and are thus preferably permeable to cells. Labels suitable for binding agents include fluorescent molecules.
- the reagents that label non-lysed cells can be detected using a variety of different techniques including, e.g., flow cytometry, fluorescence microplate readers, and fluorescence microscopy. Preferably, the presence of non-lysed cells is determined using a reporter that can be detected using flow cytometery (including, e.g., fluorescent agents such as SYBR green as well as binding molecules labelled with such fluorescent agents).
- the detection of the presence or absence of a target microorganism is determined by comparing the number of non-lysed cells between the first and second parts.
- the presence of non-lysed cells is understood to include not only the determination of whether non-lysed cells are present or absent, but also the determination of the concentration of non-lysed cells in the first and second parts and/or the determination of the relative concentration of non- lysed cells in the first part as compared to the second part.
- a reduction in the number of non-lysed cells in the sample part treated with the lysing agent indicates the presence of the target microorganism. It is within the purview of one skilled in the art to establish what constitutes a significant reduction of cells.
- the reduction of cells in the first part as compared to the second part is greater than 40, 50, 60, 70, or preferably 80%, indicating the presence of a target microorganism in a sample.
- the methods described herein comprise one or more growth steps. These steps are useful, e.g., if a greater sensitivity is needed.
- a non-selective growth step i.e., "pre-enrichment”
- pre-enrichment increases the number of microorganisms in a sample
- a selective growth step preferentially increases the number of target microorganisms in the sample. It is not uncommon for a microorganism in a sample to suffer from sublethal damage, e.g., from thermal processing of food, freezing, thawing, osmotic shock, or prolonged.
- a pre-enrichment step may be useful to restore the injured cells.
- Suitable growth media is known to one skilled in the art and includes, for example, buffered peptone water, lactose broth, tryptone soya, brilliant green, etc.
- pre-enrichment growth media is non-selective growth media.
- Samples can be inoculated on agar or liquid growth media.
- the sample is cultured for 2-24 hours, preferably for 4-8 hours, in pre-enrichment growth media.
- a selective growth step allows the growth of the targeted microorganism, while growth of competing organisms is inhibited. Examples of selection agents include antibiotics, dyes, bile salts, detergents, and other substances known to those skilled in the art.
- Selective growth for Salmonella can be carried out in, e.g., selenite cystine broth, tetrathionate broth, or Rappaport Vassiliadis soya peptone broth. It is within the purview of one skilled in the art to select suitable selective media depending on the target microorganism.
- a selective growth step may also comprise varying the growth conditions, for example by altering the temperature or CO2 levels during culture.
- the sample is cultured for 2-24 hours, preferably for 8-18 hours, in selective growth media.
- methods are provided comprising a non-selective growth step followed by a selective growth step.
- the growth steps can precede or follow steps dividing the sample, exposing the sample to a lysing agent, and exposing the sample to a binding agent.
- the order of the steps depends on the sample being tested, the microorganism being tested for, as well as the non-target microorganism in the sample. It is within the purview of one skilled in the art to determine the order of the steps in the method.
- the methods for determining the presence or absence of a target microorganism in a sample comprise culturing said sample in pre-enrichment growth media, exposing said sample to a binding agent immobilized on a solid substrate; dividing the sample into at least a first part and a second part; exposing the first part of the sample to a lytic agent, preferably a phage capable of infecting said target microorganism; providing conditions that permit the agent to lyse the microorganism, preferably conditions that permit the phage to infect and lyse the microorganism if present and culturing said samples in selective growth media; and detecting the presence of non-lysed cells in the first and second parts.
- a lytic agent preferably a phage capable of infecting said target microorganism
- the dividing of the sample into a first part and a second part may be performed before, during or after the culturing in pre-enrichment growth media and exposure to a binding agent steps.
- Example 2 describes such a preferred method.
- An additional step that may be used alone or in conjunction with additional growth steps to increase sensitivity is a step which selects for the target microorganism.
- the methods described herein further comprise a step comprising exposing the sample to a binding agent immobilized on a solid substrate.
- the binding agent preferentially binds the target microorganism over unrelated and/or related microorganisms. Unbound sample may then be removed by washing the solid support with an appropriate buffer.
- Suitable binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody, Anticalin, Microbody fibronectin-based scaffolds).
- antibodies include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies; artificial antibodies; and phage display-derived antibodies.
- binding agents against various microorganisms, as well as methods of generating specific binding agents are well known in the art.
- the specific binding agent recognizes Salmonella.
- Salmonella binding agents include RDI-TRK3S022 (Research Diagnostics) and ab31555 (AbCam).
- Binding agents may be immobilised on a solid support using conventional methods.
- Suitable solid supports include, e.g., fibers, fibrous filters, membrane filters, magnetic beads, non-magnetic beads, columns, and matrices.
- the solid support is immunomagnetic beads and an
- an immuno-separation step uses an antibody conjugated to magnetic beads.
- a selective or pre-enrichment growth step is used after the separation step.
- a selective or pre-enrichment growth step is before or after the separation step.
- the methods comprise a non-selective "pre-enrichment" growth step; exposing the sample to a binding agent immobilized on a solid substrate as described herein; dividing the sample into at least a first part and a second part either before or after the sample is exposed to a binding agent; a selective enrichment growth step;
- kits for the detection of a target microorganism in a sample comprise one or more lysing agents, preferably phages, as described herein and a reagent that labels non- lysed cells as described herein.
- Phage compositions can take the form of relatively crude lysates of bacterial cultures or highly purified virus
- Phage can be formulated in various ways for storage and shipping. Phage may be lyophilized into a dry powder and added to the growth media as a supplement during reconstitution of a powdered media or added later during the growth process if desired. Phage is also available in a liquid form or suspension. The phage may also be provided as a set of serial dilutions. Preferably, the kits further comprise a binding agent immobilized on a solid substrate as described herein.
- kits further comprise one or more of the following: filter microplates, phage diluents, buffer that provides conditions to permit the phage to infect and lyse the microorganism if present in the sample.
- the kits further comprise a microorganism susceptible to infection and lysis by the phage provided in the kit.
- the microorganism provided has a low pathogenic potential or has been modified to reduce its pathogenicity.
- a kit is provided comprising one or more bacteriophage capable of infecting one or more Salmonella serotype and a reagent that labels non-lysed cells, such as SYBR Green or a fluorescently labeled antibody specific for Salmonella.
- the kit further comprises a binding agent immobilized on a solid substrate, such as anti- Salmonella antibody conjugated immunobeads, and/or bacteria capable of being infected and lysed by the one or more bacteriophage, such as Salmonella.
- a binding agent immobilized on a solid substrate such as anti- Salmonella antibody conjugated immunobeads, and/or bacteria capable of being infected and lysed by the one or more bacteriophage, such as Salmonella.
- the methods and kits provided herein for detection of a target provide high detection sensitivity in a short amount of time without the need for lengthy cultural growth.
- the present methods and kits can provide for the detection of less than about 100, less than about 50, or 10 or less cells in a sample.
- the present methods can provide for the detection of less than about five, less than about four, less than about three, or less than about two cells in a sample.
- the methods can provide for the detection of a single cell in a sample.
- 1 CFU is detectable in 25 grams of sample.
- An exemplary embodiment for detecting a microorganism, such as Salmonella, in a sample, such as food, may include the following steps:
- an element means one element or more than one element.
- Figure 1A and IB Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Enteriditis.
- Figure 1A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control).
- Figure IB shows the analysis of the aliquot which was incubated with the phage mixture.
- Figure 2A and 2B Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Typhimurium.
- Figure 2A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control).
- Figure 2B shows the analysis of the aliquot which was incubated with the phage mixture.
- Figure 3 Results of a flow cytometric analysis of spiked minced meat samples.
- Counter refers to the bacteria count from the part of a sample in which bacteriophage has been added (+) or the part of a sample in which
- Wash medium A 0.2 ⁇ filtered PBS containing 0.05 % Tween 80 and 1% Alaska wash medium (AWM) additive
- Wash medium B 0.2 ⁇ filtered PBS containing 1 % AWM additive Peptone saline (PFZ) solution, Biokar diagnostics (prod no. BK014HA)
- Buffered peptone water (BPWT) with 0.05 % Tween 80, Biokar diagnostics (prod no. BK018HA)
- TLB Tryptone soy broth
- Oxoid Prod no. CM0129
- Salmonella selective broth Selenite brilliant green (SBG) broth, BD
- IMS Immunomagnetic separation
- Assay buffer PBS 1% (v/v) horse serum containing 0.005% (m/v) Tween 20 and 5 mM EDTA
- Salmonella enterica cultures Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
- Salmonella serovar Hadar Salmonella serovar Infantis, Salmonella serovar Anatum, Salmonella serovar Java, Salmonella serovar London, Salmonella serovar Mbandaka, Salmonella serovar Rissen, Salmonella serovar
- Salmonella phage mixture bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24. Spiking and preparation of food samples
- the selectively grown samples were diluted 1000-fold in PFZ. 10 ml of the diluted sample and 20 ⁇ of anti-Salmonella IMS beads were pipetted in a sterile 15 ml polypropylene tube. The tube was placed in a pre-heated (37°C) magnetic separator rotator (MSR) and rotated at 5 rpm for 20 min at 37°C.
- MSR magnetic separator rotator
- the liquid was removed using the vacuum manifold and the wells were washed with 200 ⁇ assay buffer.
- the IMS beads and bacteria were re-suspended in 150 ⁇ assay buffer and stained with 1.5 ⁇ SybrGreen I (diluted 1620x in assay buffer) for 15 min at room temperature.
- the liquid was removed using the vacuum manifold and both the stained bacteria and IMS beads were re-suspended in 130 ⁇ assay buffer and measured on the Beckman Coulter Cell lab Quanta SC MPL. A quantity of 2,000 IMS beads are measured as an internal reference to compare the results of different food samples.
- Minced meat samples were spiked with 13 different Salmonella spp. at approximately 10 CFU/25 g and all Salmonella spp. could be detected as exemplified for Salmonella Enteriditis and Salmonella Typhimurium in Figure 1 and Figure 2, respectively, and in Table 1.
- the darker population of dots corresponding to the IMS beads used as an internal standard is encircled in Figures 1 and 2.
- a triangle indicates the bacterial population detected in the samples.
- Table 1 demonstrates that the addition of salmonella specific bacteriophages reduces the number of bacteria by more than 85%.
- Wash medium A 0.2 ⁇ filtered PBS containing 0.05 % Tween 20 (PBST)
- Assay buffer PBS containing 1% horse serum, 5 mM EDTA and 0.005 % Tween 20
- Salmonella selective both: Selenite brilliant green (SBG) broth, BD Diagnostics ( prod no. 271510)
- IMS Immunomagnetic seperation
- Salmonella enterica cultures Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
- Salmonella phage mixture bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24.
- reaction tubes were placed in an IMS rack.
- the magnets were inserted into the rack and the reaction tubes were shaken over head by hand for 3 minutes to capture IMS beads.
- liquid inside the reaction tubes was poured out and the beads were washed with 1 ml PBST.
- the washing step was repeated twice.
- the magnet was removed from the IMS rack and IMS beads were suspended thoroughly in 100 microliter PBST.
- the re-suspended sample was split into two aliquots of 50 microliter and the aliquots were transferred to two different wells of a pre- wetted filter plate. The liquid inside the well was removed using the vacuum manifold and each well was filled with 200 microliter SBG.
Abstract
The invention relates to methods and kits for detecting a microorganism in a sample. Agents capable of lysing the microorganism are employed. Preferred lysing agents are phage. In preferred embodiments, a sample, such as a food sample, is divided into two parts. An agent capable of lysing a microorganism is added to a first part of the sample under conditions to lyse the microorganism, if present. The presence of the microorganism is detected by determining the non-lysed cells in each of the two parts. A reduction in non- lysed cells in the sample treated with a specific lysing agent is indicative of the presence of the microorganism in the sample.
Description
Title: Diagnostic methods and kits for determining the presence of a microorganism in a sample
FIELD OF THE INVENTION
The invention relates to methods and kits for detecting a microorganism in a sample. Agents, such as phage, capable of lysing the microorganism are employed.
BACKGROUND OF THE INVENTION
Contamination by microorganisms is a major cause of food and water-borne infections globally causing gastroenteritis, diarrhea, cramps, vomiting and often fever, such as that referred to as "food poisoning" to "life -threatening disease" . Illness in humans often results from the eating of undercooked meats, milk or eggs or from cross -contamination of other foods which are eaten without cooking. The most commonly recognized food borne infections are those caused by bacteria such as, Salmonella, Listeria, Campylobacter, Staphylococcus aureus and E. coli 0157:H7. For example, Salmonella is found throughout the environment, particularly in the intestinal tracts of birds, reptiles, and farm animals.
The principle bacterial pathogens that have been shown to cause human intestinal disease associated with food poisoning include Bacillus cereus, Salmonella enterica spp, Listeria monocytogenes, Vibrio paraheamolyticus, enterotoxigenic £ . coli, Campylobacter spp., Staphylococcus aureus, Yersinia enterocolitica, and Clostridium perfringens. Conventional methods for the detection of contamination employ non-selective or selective culturing, or enrichment, followed by plating the cultures on selective media for verification of suspect colonies. This approach is time consuming and can take several days before results are obtained.
Improved diagnostic methods and kits are therefore needed for the detection of microorganism in a sample, such as a bacterial contaminant in food, water, environmental, medical, agricultural, veterinary, pharmaceutical or industrial fermentation preparations.
SUMMARY OF THE INVENTION
The present disclosure provides rapid and highly sensitive methods and kits for the detection of microorganisms in a sample. In one aspect, the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to an agent capable of lysing said target microorganism; providing conditions that permit the agent to lyse the microorganism, if present; and detecting the presence of non-lysed cells in the first and second parts. Preferably, said agent is a phage and the method comprises the steps of exposing the first part of the sample to a phage capable of infecting said target microorganism and providing conditions that permit the phage to infect and lyse the microorganism.
In one aspect, the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism, if present;
collecting a cell free extract from the first part and a cell free extract from the second part; adding a microorganism susceptible to phage infection to said cell free extracts; providing conditions that permit the phage, if present, to infect and lyse the added microorganism; detecting the presence of non-lysed cells in the first and second parts.
Preferably, the microorganism is a bacterium and the phage is a
bacteriophage.
Preferably, a decrease in cells in the first part in comparison to the second part indicates the presence of the microorganism in the sample.
Preferably, the methods further comprise exposing the sample to a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism such that the microorganism becomes associated with the solid substrate. Preferably, the solid support is
immunomagnetic beads. Preferably, the binding agent is an antibody.
Preferably, the methods further comprise one or more growth steps.
Preferably, one or more of the growths steps is a selective growth step. In some embodiments, the methods further comprise at least one pre-enrichment growth step and/or at least one selective growth step, preferably the selective growth step follows the pre-enrichment step.
In some embodiments, the methods further comprise exposing both first and second parts to a reagent that labels non-lysed cells and detecting said reagent to determine the presence of non-lysed cells in the first and second parts.
Preferably, the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labelled binding agent, such as an antibody, capable of binding the microorganism. Preferably, the reagent that labels non-lysed cells is detected using flow cytometry.
In some embodiments of the methods, the target microorganism is Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22-
like, T7-like, epsilonl5, KS7, Felix 01 bacteriophage, or a combination thereof. Preferably, the phage is Felix 01.
In one aspect, the disclosure provides diagnostic kits for the detection of a target microorganism in a sample comprising an agent that specifically lyses a target microorganism, preferably a phage, and a reagent that labels non-lysed cells. In some embodiments, the kit further comprises a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism if present in the sample such that the microorganism becomes associated with the solid substrate. Preferably, the solid support is immunomagnetic beads. Preferably, the binding agent is an antibody. In some embodiments, the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labeled binding agent, such as an antibody, capable of binding the microorganism. In some embodiments, the kit further comprises a microorganism susceptible to lysis by the lysing agent, e.g. a microorganism susceptible to infection by said phage. In some embodiments, the kit further comprises further a microorganism that is not susceptible to infection by said phage. Preferably, the microorganism is a bacterium and the phage is a bacteriophage.
In some embodiments, the target microorganism detected by the kit is
Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22-like, T7-like, epsilonl5, KS7, Felix 01 virus, or a combination thereof.
Preferably, the microorganism is a bacterium and the phage is a
bacteriophage.
DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS
Attempts have been made to improve upon classical bacterial detection methods using bacteriophage infection and/or amplification. Phages are viruses that have evolved in nature to use microorganism as a means of replicating themselves. A bacteriophage, for example, 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 bacteria releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for phage infection of bacteria, phage multiplication
(amplification) in the bacteria, and release of the progeny phage after lysis, can take as little as an hour or less depending on the phage, the bacteria, and the environmental conditions. Microbiologists have isolated and characterized over 5,000 phage species, including many that specifically target bacteria at the species or even the strain level.
Methods for bacterial detection using bacteriophage which are genetically modified to express a reporter molecule are described in, e.g., U. S. Patents 4,861, 709 and 5,824, 468. These methods suffer from the disadvantage that a suitable bacteriophage must be genetically modified in order to detect a pathogen.
Other described methods detect bacterial intracellular components after phage induced lysis. U. S. Patent 5,888, 725 describes a method utilizing unmodified, highly specific lytic phages to infect target bacteria in a sample. Phage-induced lysis releases certain nucleotides from the bacterial cell such as ATP that can be detected using known techniques. U.S. Patent 6,436,661 describes a method whereby a phage is used to infect and lyse a target bacterium in a sample releasing intracellular enzymes, which react in turn with an immobilized enzyme substrate, thereby producing a detectable signal. Alternative methods
detect the bacteriophage amplified in a target microorganism.
WO 2006/083292 describes the detection of bacteriophage protein or nucleic acid. While these methods have the advantage of using unmodified phage, the sensitivity of the assays is limited as the concentration of detected markers (nucleotides or enzymes) is directly proportional to the concentration of target bacteria in the sample. These methods also often have a relatively high background level due to the presence of the detected components in the sample. For example, meat also contains enzymes and nucleotides. The present disclosure provides methods and kits for detecting
microorganisms utilizing the sensitivity and specificity of phages without the costly and time-consuming requirement that the phages be genetically modified. In one aspect, the disclosure provides methods for detecting the presence or absence of a microorganism in a sample comprising dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present; and detecting the presence of non-lysed cells in the first and second parts.
These preferred methods of the invention detect cells which have not been lysed by the previous step of phage infection. Other methods have been described previously which require multiple steps to recover the phages after infection and subsequently infect healthy microorganisms (see, e.g., Favrin et al. Applied and Environmental Microbiology 67:217-224). Such methods use healthy microorganisms as a detector of amplified phage. In contrast, the preferred methods of the invention directly detect the presence of target microorganisms in a sample based on their susceptibility to a specific lysing agent, e.g., a phage.
The present disclosure demonstrates in the Examples that specific lysis of target microorganisms by an agent permits the identification of said
microorganism in a sample. Accordingly, the present disclosure provides that any agent capable of specifically lysing a target microorganism may be used in the methods and kits disclosed herein. Preferred agents are the phages disclosed herein. Other lytic agents that specifically lyse a target
microorganism include, e.g., antibiotics and bactericides. In order to increase their specificity for a target microorganism, said agents may be coupled to, e.g., antibodies that specifically recognize the target microorganism.
In one aspect of the disclosure, the methods described herein comprise additional steps that may increase the sensitivity of the assay. In some embodiments, methods are provided for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present;
collecting a cell free extract from the first part, preferably after the phage has gone through at least one multiplication cycle, and a cell free extract from the second part; adding a microorganism susceptible to phage infection to said cell free extracts; providing conditions that permit the phage, if present, to infect and lyse the added microorganism; and detecting the presence of non-lysed cells in the first and second parts. While not wishing to be bound by theory, it is thought that the agent, preferably a phage will lyse the microorganism if present, thereby decreasing the number of intact cells in the first part of the sample in comparison to the second part of the sample. In some embodiments, the presence of a
microorganism in a sample is indicated by a greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%,
preferably greater than 85%, or greater than 90% reduction in cell number in the first part of the sample, i.e., agent treated, as compared to the untreated part of the sample. As used herein, microorganisms refer to bacteria, mycoplasmas, and other microscopic living organisms. Preferably the microorganisms are bacteria.
Target bacteria contemplated by the present methods and kits include, but are not limited to, bacterial cells that are food, water, or environmental
contaminants, pathogens of agricultural, medical or veterinary significance, and bacterial cells useful in pharmaceutical, industrial or commercial processes. It will be understood by those skilled in the art that the term
"detection of bacteria" relates to a single species, isolate, serovar or strain as well as groups or families of organisms.
Bacteria include, but are not limited to, Acinetobacter spp., Actinomyces spp., Bacillus spp. (e.g., Bacillus anthracis, Bacillus subtilis, Bacillus cereus), Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucellae (e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella canis),
Costridium spp. (e.g., Clostridium difficile, Clostridium perfringens)
Corynebacterium diphtheriae, Campylobacter jejuni, Chlamydia pneumoniae, Enterococci (e.g., Enterococcus faecalis), Escherichia coli, Eubacterium alactolyticum, Enterobacter spp. (e.g., Enterobacter cloacae), Francisella tularensis, Flavobacterium meningosepticum, Helicobacter pylori, Klebsiellae (e.g., Klebsiella pneumoniae), Legionella spp. (e.g., Legionella pneumophilia), Leptospira interrogans, Listeria monocytogenes, Mycobacterium spp. (e.g. Mycobacterium avium avium, Mycobacterium avium paratuberculosis), Moraxella catarrhalis, Moraxella lacunata, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus, (e.g., Proteus mirabilis, Proteus vulgaris), Pseudomonas spp., Providencia alcalifaciens, Providencia stuartii, Providencia rettgeri,
Rickettsia prowazekii, Salmonellae, Serratia spp., Staphylococci (e.g.,
Staphylococcus aureus, Staphylococcus epidermidis), Shigella spp. (e.g., Shigella dysenteriae), Spirillum minus, Streptococci, Treponema pallidum, Ureaplasma urealyticum, Vibrio spp. (e.g., Vibrio cholerae, Vibrio vulnificus), Xanthomonas maltophilia, and Yersinsia spp (e.g., Yersinia pestis).
In some embodiments, bacteria are selected from Salmonella; E. coli, preferably E. coli 0157:H7; Listeria, preferably L. monocytogenes; Legionella and Campylobacter.
The genus Salmonella comprises two species, Salmonella bongori and
Salmonella enterica. Salmonella enterica is divided into serovars or serotypes, based on somatic O-antigens, flagellar (H) antigens, and surface antigens. There are at least 1500 food-borne Salmonella serovars identified so far.
Salmonella serotypes detectable using the methods and kits described herein include, for example, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Java, Salmonella enterica serovar Infantis, Salmonella enterica serovar Virchow, Salmonella enterica serovar Mbandaka. Salmonella enterica serovar Livingstone,
Salmonella enterica serovar Senftenberg, Salmonella enterica serovar Hadar, Salmonella enterica serovar London, Salmonella enterica serovar Rissen, Salmonella enterica serovar Anatum, Salmonella enterica serovar Bredeney, and Salmonella enterica serovar Derby. While all Salmonella enterica serotypes are considered potentially pathogenic to humans, only a few are pathogenic to animals. They are often the inhabitants of healthy poultry and pigs, which serve as reservoirs for human transmission. Some serotypes do demonstrate host specificity. The identification of a particular subspecies or serotype can therefore provide information regarding, for example, the contamination route.
In one aspect, the disclosure provides methods as described herein for detecting the presence or absence of one or more bacterial serotypes in a sample by using one or more bacteriophages that are capable of distinguishing between one or more serotypes. As used herein, serotype and serovar refers to bacteria within the same species that can be distinguished on the basis of their surface antigenic properties.
The methods provided are useful for determining the presence or absence of microorganisms in a wide variety of sample material. Samples include, e.g., various food products, water, industrial or agricultural products, biological samples such as urine, feces, sputum, blood or tissue samples, and may be solid or liquid samples.
Phages, preferably lytic phages, are employed in the methods and kits described herein due to their high degree of specificity when infecting a microorganism. Lytic phages also includes phages which are normally non- lytic but have been modified to induce lysis in the host microorganism. A method for the modification of such phages is described in U.S. Patent
Publication 2003/0180319. As used herein, a phage capable of infecting said target microorganism refers to the ability of the phage to infect and replicate within the host and ultimately destroy the cell while releasing numerous phage progeny into the medium. The term phage includes, for example, bacteriophages,
mycobacteriophage, and mycoplasma phage. Preferably, the phage is a bacteriophage.
The specificity of a phage for its host is determined at two levels. The first level of control involves the interaction of phage components with
complementary elements on the host cell surface, which determines the ability of the phage to bind to the cell and inject its DNA. The second level of control
over specificity is during the events occurring within the host cell, after injection of the phage DNA. In some embodiments, a phage is specific for a given host when it is capable of infecting the given host and does not infect cells of another species or strain.
As used herein, conditions that permit the agent to lyse the microorganism or particulary conditions that permit the phage to infect and lyse the
microorganism refers to conditions associated with, for example, time, temperature, and suitable buffering conditions that are well-known to one skilled in the art. (See for example Sands JA, et al., 1974 Biochimica et
Biophysica Acta (BBA) 373:277-285; Phage Ecology, edited by S.M. Goyal, CP. Gerba and G. Bitton, John Wiley and Sons, 1987, New York, USA; and
Example 1 of the present disclosure) The methods and kits provided herein can also be used to determine the presence or absence of more than one microorganism or more than one bacterial serotype in a single assay by using one or more phages. In some embodiments, a single phage can recognize, for example, more than one bacterial serotype. In some embodiments, a combination of phages is used that recognize different host species, different host strains, or preferably different host serotypes.
Phage useful in the methods and kits described herein also may include genetically modified or recombinant phage that have been altered to increase binding affinity, infectivity, burst size, multiplicity of infection or lytic ability for the microorganism to be detected. Preferably, the phages useful in the methods and kits described herein have not been genetically modified to express a report molecule. In some embodiments the phage is not genetically modified.
A wide variety of bacteriophages are available for any given bacterial cell, for example, from the American Type Culture Collection (ATCC, P.O. Box 1549 Manassas, Va., USA) or by isolation from natural sources that harbor the host cells. A list of phage types is published as the Catalogue of Bacteria &
Bacteriophages. (American Type Culture Collection, Rockville, Md. 1989). Over 20,000 strains of bacteria (with 7,000 being Salmonella) have been evaluated for phage identification (He and Pan, 1992, J Clin Microbiol 30(3): 590-4).
Salmonella specific phages useful for the methods and kits described herein include, e.g., b , Beccles , CT , d , Dundee , f , Fels 2 , GI , GUI , GVI , GVIII , k , K , i , j , L , 01 , (syn= O-l) , (syn= 01) , (syn= O-I) , (syn= 7) , 02 , 03 , P3 , P9a , P10 , Sab3 , Sab5 , Sanl5 , Sanl7 , SI , Taunton , Vil , (syn= Vil) , 9 , and NN-Salmonella (1), P27-like, P2-like, lambdoid, P22-like, T7-like, epsilonl5, KS7, Ent, MS24, and Felix 01. Preferably, the phage is Felix 01. Additional Salmonella bacteriophages and method for isolating phages are described in PCT Publication WO2005/024005 and Capparelli R et al., J Infect Dis. 2010 Jan 1;201(1):52-61 A skilled person is aware of additional
Salmonella phages which are described, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
Additional bacteriophages include, but are not limited to
Actinoplanes/Micromonospora phages (Ap3, Ap4, Mml, Mm3, Mm4, Mm5, phiUW 51); Amycolatopsis phages (W2, W4, W7, Wll); Bacillus phages (GA-1, Phi 29, SP.beta.); Campylobacter phages (e.g., NTCC 12669 , NTCC 12670 , NTCC12671 , NTCC12672 , NTCC12673 , NTCC12674 , NTCC12675 ,
NTCC12676 , NTCC12677 , NTCC12678 , NTCC12679 , NTCC12680 ,
NTCC12681 , NTCC12682 , NTCC12683 , NTCC12684); Cellulomonas phages (02, 03, 05, 06, 08, Oil, 013); Escherichia phages (lambda, M13, M13, mpl8, MS2, Mu, PI, PhiX174, Q.beta, R17, Tl, T2, T3, T4, T5, T6, T7, U3); Lactococcus phages (P001, P008); Listeria phages (A005 , A006 , A020 , A500 ,
A502 , A511 , A118 , Α620 , Α640 , B012 , B021 , Β024 , Β025 , Β035 , B051 , Β053 , Β054 , Β055 , Β056 , B101 , B110 , Β545 , Β604 , Β653 , C707 , D441 , HS047 , H10G , Η8/73 , H19 , H21 , Η43 , Η46 , H107 , H108 , H110 , H163/84 , H312 , Η340 , Η387 , H391/73 , Η684/74 , Η924Α , PSA , U153 , MLUP5 , (syn= Ρ35) , 00241 , 00611 , 02971A , 02971C , 5/476 , 5/911 , 5/939 , 5/11302 , 5/11605 , 5/11704 , 184 , 575 , 633 , 699/694 , 744 , 900 , 1090 , 1317 , 1444 , 1652 , 1806 , 1807 , 1921/959 , 1921/11367 , 1921/11500 , 1921/11566 ,
1921/12460 , 1921/12582 , 1967 , 2389 , 2425 , 2671 , 2685 , 3274 , 3550 , 3551 , 3552 , 4276 , 4277 , 4292 , 4477 , 5337 , 5348/11363 , 5348/11646 , 5348/12430 , 5348/12434 , 10072 , 11355C , 11711A , 12029 , 12981 , 13441 , 90666 , 90816 , 93253 , 907515 , 910716), Methanothermobacter phage (psi Μ2 (.PSI.M2)); Nocardia/Rhodococcus/Gordonia phages (N4, N5, N13, N18, N24, N26, N36); Nocardioides phages (XI, X3, X5, X6, X10, X24); Nocardiopsis phages (D3, D4); Promicromonospora phages (PI, P2, P3, P4); Pseudomonas phages (Pspl, Psp2, Psp3, Psp4, Psp5); Pseudonocardia phage (W3); Saccharomonospora phages (Tml, Tm2, Tm3); Saccharopolyspora phages (Mpl, Mp2);
Saccharothrix phage (Wl); Sporichthya phage (Spl); Streptomyces phages (P23, P26, SI, S2, S3, S4, S6, S7, SH10, phi A. streptomycini III, phi8238, phiC31); Terrabacter phages (Tbl, Tb2); Tsukamurella phage (Tsl). Other suitable phages are known to one of skill in the art and may be found, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
Other bacteriophages with a high level of specificity can be developed by screening samples using methods described in U.S. Pat. No. 6,322,783, which is incorporated herein by reference. Phages specific for particular bacteria can also be selected using routine techniques in the laboratory due to the ability of the phage to rapidly mutate, thereby producing host range mutants.
The terms "lysed cell" and "non-lysed cell" are well-understood to one of skill in the art. For example, a non-lysed cell has a cell wall significantly intact to
allow its detection by various assays known to one of skill in the art.
Conventional cell culturing employs adding various dilutions of, e.g., bacteria culture to agar plates and counting the number of bacterial colonies that form. As alternatives, instruments have been developed using various principles of detection including infrared or fluorescence spectroscopy, bioluminescence, and flow cytometry (Bird et al. (1989) Rapid Salmonella Detection by a
Combination of Conductance and Immunological Techniques; Blackwell Sci. Publications: Oxford, Vol. 25; Fenselau, Ed. (1994) Mass Spectrometry for the Characterization of Microorganisms Washington D.C., Vol. 240; Lloyd, Ed. (1993) Flow Cytometry in Microbiology; Springer-Verlag London Limited: Germany; Perez, et al. (1998) Anal. Chem. 70:2380-2386; Wyatt (1995) Food Agri. Immunol. 7:55-65). Among these, the primary physical/chemical methods of bacterial detection are those which involve the detection of some naturally occurring component of the bacterium. Other devices and methods for detecting microorganisms are provided in U.S. Pat. Nos. 5,094,955; 6,777,226; 6, 197,577; 5,976,827; and 5,912, 115. In general, these devices rely on the use of a single sensor (e.g., pH or carbon dioxide indicator) in a layer adjacent to a layer of growth medium for detecting the presence of a bacterium. Preferably, the presence of non-lysed cells is detected using a reagent that labels non-lysed cells. Suitable reagents include reagents which are permeable to cells, e.g., SYBR Green, oxazole yellow, thiazole orange, ethidium bromide, fluorescein diacetate,and PicoGreen. Other reagents for labelling non-lysed cells include labelled-binding agents capable of binding the microorganism. The binding agent may preferentially bind the target microorganism over non- target microorganisms or may bind microorganisms indiscriminately. Binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody,
Anticalin, Microbody fibronectin-based scaffolds). As used herein, antibodies
include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies, artificial antibodies, and phage display-derived antibodies. Binding agents against various microorganisms, as well as methods of generating binding agents are well known in the art. The binding agents may recognize, for example,
transmembrane proteins, outer membrane proteins, or components of the cell membrane/cell wall. Suitable binding agents include, e.g., anti-lipid A antibodies (AbCam # ab20001). In some embodiments, the binding agents recognize intracellular components and are thus preferably permeable to cells. Labels suitable for binding agents include fluorescent molecules. The reagents that label non-lysed cells can be detected using a variety of different techniques including, e.g., flow cytometry, fluorescence microplate readers, and fluorescence microscopy. Preferably, the presence of non-lysed cells is determined using a reporter that can be detected using flow cytometery (including, e.g., fluorescent agents such as SYBR green as well as binding molecules labelled with such fluorescent agents). Preferably, the detection of the presence or absence of a target microorganism is determined by comparing the number of non-lysed cells between the first and second parts. The presence of non-lysed cells is understood to include not only the determination of whether non-lysed cells are present or absent, but also the determination of the concentration of non-lysed cells in the first and second parts and/or the determination of the relative concentration of non- lysed cells in the first part as compared to the second part. A reduction in the number of non-lysed cells in the sample part treated with the lysing agent indicates the presence of the target microorganism. It is within the purview of one skilled in the art to establish what constitutes a significant reduction of cells. In some embodiments, the reduction of cells in the first part as compared
to the second part is greater than 40, 50, 60, 70, or preferably 80%, indicating the presence of a target microorganism in a sample.
In some embodiments, the methods described herein comprise one or more growth steps. These steps are useful, e.g., if a greater sensitivity is needed. A non-selective growth step, i.e., "pre-enrichment", increases the number of microorganisms in a sample, while a selective growth step preferentially increases the number of target microorganisms in the sample. It is not uncommon for a microorganism in a sample to suffer from sublethal damage, e.g., from thermal processing of food, freezing, thawing, osmotic shock, or prolonged. A pre-enrichment step may be useful to restore the injured cells. Suitable growth media is known to one skilled in the art and includes, for example, buffered peptone water, lactose broth, tryptone soya, brilliant green, etc. Preferably, pre-enrichment growth media is non-selective growth media. Samples can be inoculated on agar or liquid growth media. Preferably, the sample is cultured for 2-24 hours, preferably for 4-8 hours, in pre-enrichment growth media. A selective growth step allows the growth of the targeted microorganism, while growth of competing organisms is inhibited. Examples of selection agents include antibiotics, dyes, bile salts, detergents, and other substances known to those skilled in the art. Selective growth for Salmonella can be carried out in, e.g., selenite cystine broth, tetrathionate broth, or Rappaport Vassiliadis soya peptone broth. It is within the purview of one skilled in the art to select suitable selective media depending on the target microorganism. A selective growth step may also comprise varying the growth conditions, for example by altering the temperature or CO2 levels during culture. Preferably, the sample is cultured for 2-24 hours, preferably for 8-18 hours, in selective growth media.
Preferably, methods are provided comprising a non-selective growth step followed by a selective growth step. As described in more detail later and exemplified in the examples, the growth steps can precede or follow steps dividing the sample, exposing the sample to a lysing agent, and exposing the sample to a binding agent. The order of the steps depends on the sample being tested, the microorganism being tested for, as well as the non-target microorganism in the sample. It is within the purview of one skilled in the art to determine the order of the steps in the method.
Preferably, the methods for determining the presence or absence of a target microorganism in a sample comprise culturing said sample in pre-enrichment growth media, exposing said sample to a binding agent immobilized on a solid substrate; dividing the sample into at least a first part and a second part; exposing the first part of the sample to a lytic agent, preferably a phage capable of infecting said target microorganism; providing conditions that permit the agent to lyse the microorganism, preferably conditions that permit the phage to infect and lyse the microorganism if present and culturing said samples in selective growth media; and detecting the presence of non-lysed cells in the first and second parts. It is clear to a skilled person that the dividing of the sample into a first part and a second part may be performed before, during or after the culturing in pre-enrichment growth media and exposure to a binding agent steps. Example 2 describes such a preferred method. An additional step that may be used alone or in conjunction with additional growth steps to increase sensitivity is a step which selects for the target microorganism. Preferably, the methods described herein further comprise a step comprising exposing the sample to a binding agent immobilized on a solid substrate. Preferably, the binding agent preferentially binds the target microorganism over unrelated and/or related microorganisms. Unbound
sample may then be removed by washing the solid support with an appropriate buffer. By separating the captured target microorganism from other
microorganisms, the background signal may be reduced in the assay. Suitable binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody, Anticalin, Microbody fibronectin-based scaffolds). As used herein, antibodies include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies; artificial antibodies; and phage display-derived antibodies. Binding agents against various microorganisms, as well as methods of generating specific binding agents are well known in the art.
In some embodiments, the specific binding agent recognizes Salmonella.
Commercially available Salmonella binding agents include RDI-TRK3S022 (Research Diagnostics) and ab31555 (AbCam).
Binding agents may be immobilised on a solid support using conventional methods. Suitable solid supports include, e.g., fibers, fibrous filters, membrane filters, magnetic beads, non-magnetic beads, columns, and matrices. In some embodiments, the solid support is immunomagnetic beads and an
immumomagnetic separation (IMS) technique is used to capture and concentrate the target microorganism. Preferably, an immuno-separation step uses an antibody conjugated to magnetic beads. In some embodiments, a selective or pre-enrichment growth step is used after the separation step. In some embodiments, a selective or pre-enrichment growth step is before or after the separation step.
In particularly preferred methods of the disclosure, the methods comprise a non-selective "pre-enrichment" growth step; exposing the sample to a binding agent immobilized on a solid substrate as described herein; dividing the sample into at least a first part and a second part either before or after the sample is exposed to a binding agent; a selective enrichment growth step;
exposing said first part of the sample to a lysing agent as described herein; and detecting the presence of non-lysed cells in the first and second parts.
In one aspect of the disclosure, diagnostic kits for the detection of a target microorganism in a sample are provided. The kits comprise one or more lysing agents, preferably phages, as described herein and a reagent that labels non- lysed cells as described herein. Phage compositions can take the form of relatively crude lysates of bacterial cultures or highly purified virus
preparations. Phage can be formulated in various ways for storage and shipping. Phage may be lyophilized into a dry powder and added to the growth media as a supplement during reconstitution of a powdered media or added later during the growth process if desired. Phage is also available in a liquid form or suspension. The phage may also be provided as a set of serial dilutions. Preferably, the kits further comprise a binding agent immobilized on a solid substrate as described herein.
In some embodiments, the kits further comprise one or more of the following: filter microplates, phage diluents, buffer that provides conditions to permit the phage to infect and lyse the microorganism if present in the sample. In preferred embodiments, the kits further comprise a microorganism susceptible to infection and lysis by the phage provided in the kit. Preferably, the microorganism provided has a low pathogenic potential or has been modified to reduce its pathogenicity.
In an exemplary embodiment, a kit is provided comprising one or more bacteriophage capable of infecting one or more Salmonella serotype and a reagent that labels non-lysed cells, such as SYBR Green or a fluorescently labeled antibody specific for Salmonella. In some embodiments, the kit further comprises a binding agent immobilized on a solid substrate, such as anti- Salmonella antibody conjugated immunobeads, and/or bacteria capable of being infected and lysed by the one or more bacteriophage, such as Salmonella.
The methods and kits provided herein for detection of a target provide high detection sensitivity in a short amount of time without the need for lengthy cultural growth. For example, the present methods and kits can provide for the detection of less than about 100, less than about 50, or 10 or less cells in a sample. Preferably the present methods can provide for the detection of less than about five, less than about four, less than about three, or less than about two cells in a sample. Most preferably, the methods can provide for the detection of a single cell in a sample. In some embodiments, 1 CFU is detectable in 25 grams of sample.
The methods and kits described above are widely applicable to the detection of a range of microorganisms from a wide variety of samples. An exemplary embodiment for detecting a microorganism, such as Salmonella, in a sample, such as food, may include the following steps:
(i) starting with, e.g., a test food sample, culture the sample in a liquid culture broth, preferably for 2-24 h, preferably with shaking;
(ii) optionally, replace media with media selective for the target microorganism and culture for another 2-24 hours;
(iii) optionally, capture and concentrate microorganism from the broth using immunomagnetic particles with an immobilized binding agent, such as an antibody;
(iv) divide sample into at least two parts, this step can follow any of steps (i)- (iii);
(v) add phage to one part of sample under conditions to permit infection and lysis;
(vi) add a reagent, such as SYBR Green, that detects non-lysed cells to two parts of the sample;
(vii) detect reagent in the two parts of the sample, such as, e.g, using flow cytometry. As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A and IB: Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Enteriditis. Figure 1A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control). Figure IB shows the analysis of the aliquot which was incubated with the phage mixture.
Figure 2A and 2B: Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Typhimurium. Figure 2A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control). Figure 2B shows the analysis of the aliquot which was incubated with the phage mixture. Figure 3: Results of a flow cytometric analysis of spiked minced meat samples. "Count" refers to the bacteria count from the part of a sample in which bacteriophage has been added (+) or the part of a sample in which
bacteriophage was not added (-).
EXAMPLES
Example 1
Material and methods:
Wash medium A (WMA): 0.2 μηι filtered PBS containing 0.05 % Tween 80 and 1% Alaska wash medium (AWM) additive
Wash medium B (WMB): 0.2 μηι filtered PBS containing 1 % AWM additive Peptone saline (PFZ) solution, Biokar diagnostics (prod no. BK014HA)
Buffered peptone water (BPWT) with 0.05 % Tween 80, Biokar diagnostics (prod no. BK018HA)
Tryptone soy broth (TSB), Oxoid (Prod no. CM0129)
Salmonella selective broth: Selenite brilliant green (SBG) broth, BD
diagnostics (prod no. 271510)
Immunomagnetic separation (IMS) beads, Invitrogen dynabeads® anti- salmonella (Cat no.71002)
Assay buffer: PBS 1% (v/v) horse serum containing 0.005% (m/v) Tween 20 and 5 mM EDTA
Pall Acroprep™ 96, 0.2 μηι filter microplate
SybrGreen I (stock solution in DMSO), Sigma Aldrich (prod no.S9430)
Salmonella enterica cultures: Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
Salmonella serovar Hadar, Salmonella serovar Infantis, Salmonella serovar Anatum, Salmonella serovar Java, Salmonella serovar London, Salmonella serovar Mbandaka, Salmonella serovar Rissen, Salmonella serovar
Senftenberg, Salmonella serovar Typhimurium and Salmonella serovar Virchow were grown overnight in TSB at 37°C.
Salmonella phage mixture: bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24.
Spiking and preparation of food samples
Overnight cultures of Salmonella were diluted serially (1: 10) in pre-warmed (37 °C) PFZ. 25 g of minced meat samples were weighed in a stomacher back and spiked with 1 ml of the 10"8 dilution of the respective Salmonella ssp. listed above. To simulate a cold shock, the spiked minced meat samples were stored over 48 hours at 4°C. After the incubation at 4°C, 225 ml of BPWT were added to the minced meat and the food sample was homogenized for 30 seconds using a Stomacher circulator. Subsequently, the homogenized food sample was incubated for 6 hours at 37 °C. After this non-selective growth, 1 ml of the pre-enriched sample was transferred to 9 ml of SBG broth and incubated overnight at 37°C (selective growth step).
IMS and flow cytometric assay
The selectively grown samples were diluted 1000-fold in PFZ. 10 ml of the diluted sample and 20 μΐ of anti-Salmonella IMS beads were pipetted in a sterile 15 ml polypropylene tube. The tube was placed in a pre-heated (37°C) magnetic separator rotator (MSR) and rotated at 5 rpm for 20 min at 37°C.
After the 20 min incubation period, magnets were inserted into the racks of the MSR and the tubes were rotated at 5 rpm for 5 min at 37°C to separate IMS beads. Racks are removed from the MSR and the liquid was removed using a vacuboy (Integra biosciences). The IMS beads were washed by adding 10 ml of WMA and rotating the tubes again at 5 rpm for 5 min at 37°C. Racks were removed from the MSR and the liquid was removed using the vacuboy. To suspend the IMS beads, the magnets were removed from the racks and
0.5 ml of WMB were added to each sample tube. The IMS beads were vortexed and, immediately, two 240 μΐ aliquots of each sample were transferred to two wells of a pre-wetted filter microplate. The liquid was removed using the vacuum manifold calibrated and the IMS beads are washed with 200 μΐ WMB.
Growth medium (TSB) was added to one of the two aliquots (phage diluent in Figures 1A and 2A). TSB containing a mix of three different Salmonella specific bacteriophages was added to the other aliquot to lyse Salmonella cells and the plate was incubated for 2.0-2.5 hours at 37°C (phage mixture in Figures IB and 2B).
After this incubation step, the liquid was removed using the vacuum manifold and the wells were washed with 200 μΐ assay buffer. The IMS beads and bacteria were re-suspended in 150 μΐ assay buffer and stained with 1.5 μΐ SybrGreen I (diluted 1620x in assay buffer) for 15 min at room temperature.
The liquid was removed using the vacuum manifold and both the stained bacteria and IMS beads were re-suspended in 130 μΐ assay buffer and measured on the Beckman Coulter Cell lab Quanta SC MPL. A quantity of 2,000 IMS beads are measured as an internal reference to compare the results of different food samples.
Evaluation of results:
Minced meat samples were spiked with 13 different Salmonella spp. at approximately 10 CFU/25 g and all Salmonella spp. could be detected as exemplified for Salmonella Enteriditis and Salmonella Typhimurium in Figure 1 and Figure 2, respectively, and in Table 1. The darker population of dots corresponding to the IMS beads used as an internal standard is encircled in Figures 1 and 2. A triangle indicates the bacterial population detected in the samples. Table 1 demonstrates that the addition of salmonella specific bacteriophages reduces the number of bacteria by more than 85%.
The experiment was repeated using the method as described above to compare the results obtained from the phage mixture with the use of Felix 01 virus alone. As demonstrated in Figure 3, similar results were achieved using a single phage or a mixture of phage.
Example 2:
Material and methods
Buffered peptone water (BPW) supplemented with 0.05% Tween 80, Biokar diagnostics (prod no. BK018HA)
Peptone-buffered saline (PFZ) solution, Biokar diagnostics (prod no.
BK014HA)
Tryptone soy broth (TSB), Oxoid (prod no. CM0129)
Wash medium A: 0.2 μιη filtered PBS containing 0.05 % Tween 20 (PBST) Assay buffer: PBS containing 1% horse serum, 5 mM EDTA and 0.005 % Tween 20
Salmonella selective both: Selenite brilliant green (SBG) broth, BD Diagnostics ( prod no. 271510)
Immunomagnetic seperation (IMS) beads, Invitrogen dynabeads® anti- Salmonella (prod no. 71002)
Pall Acroprep™ 96, 0.2 micrometer filter plate
SybrGreen I (stock solution in DMSO), Sigma Aldrich (prod no. S9430)
Salmonella enterica cultures: Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
Salmonella serovar Hadar, Salmonella serovar Infantis, Salmonella serovar Java, Salmonella serovar Livingstone, Salmonella serovar London, Salmonella serovar Mbandaka, Salmonella serovar Panama, Salmonella serovar Rissen, Salmonella serovar Senftenberg, Salmonella serovar Typhimurium and Salmonella serovar Virchow were grown overnight in TSB at 37°C.
Salmonella phage mixture: bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24.
As negative control samples, overnight cultures of E. coli, Proteus spp. and Enterobacter spp. were diluted as described above for Salmonella and the diluted samples were used to spike minced meat samples. Spiking and preparation of food samples
Overnight cultures of Salmonella were diluted serially (1: 10) in pre-warmed (37 °C) PFZ. 25 g of minced meat samples were weighed in a stomacher back and spiked with 1 ml of the 10"8 dilution of the respective Salmonella ssp. listed above. To simulate a cold shock, the spiked minced meat samples were stored over 48 hours at 4°C. After incubation at 4°C, 225 ml of BPWT were
added to the minced meat and the food sample was homogenized for 30 seconds using a Stomacher circulator. Subsequently, the homogenized food sample was incubated overnight at 37°C. IMS and flow cytometric assay
After this non-selective growth step, over night cultures were diluted 100-fold in PFZ and 1 ml of the diluted samples were incubated with 20 microliter IMS beads in a sterile 1 ml polypropylene reaction tube. IMS beads and diluted sample were incubated for 10 minutes on an orbital shaker at room
temperature. Subsequently, reaction tubes were placed in an IMS rack. The magnets were inserted into the rack and the reaction tubes were shaken over head by hand for 3 minutes to capture IMS beads. After capturing of IMS beads, liquid inside the reaction tubes was poured out and the beads were washed with 1 ml PBST. The washing step was repeated twice. The magnet was removed from the IMS rack and IMS beads were suspended thoroughly in 100 microliter PBST. The re-suspended sample was split into two aliquots of 50 microliter and the aliquots were transferred to two different wells of a pre- wetted filter plate. The liquid inside the well was removed using the vacuum manifold and each well was filled with 200 microliter SBG. One aliquot of the sample was supplemented with 60 microliter of a bacteriophage solution and the other aliquot of the sample was supplemented with 60 microliter of phage diluent, e.g. PBST. The filter plate was incubated for 4 hours on a micro plate shaker at 300 rpm and 37°C. After this incubation step, the liquid was removed using the vacuum manifold and the wells were washed with 200 μΐ assay buffer. The IMS beads and bacteria were re-suspended in 150 μΐ assay buffer and stained with 50 μΐ SybrGreen I (diluted 20,000x in assay buffer) for 15 min at room temperature. The liquid was removed using the vacuum manifold and both the stained bacteria and IMS beads were re-suspended in 130 μΐ assay buffer and 5 microliter of a sample were measured on an Accuri C6 flow cytometer.
Evaluation of results
To evaluate the samples, the number of bacteria detected in the two aliquots were determined and compared. Table 2 confirms that that the addition of Salmonella specific bacteriophages reduces the number of bacteria by more than 80%.
Claims
1. A method for determining the presence or absence of a target microorganism in a sample comprising:
dividing the sample into at least a first part and a second part;
exposing the first part of the sample to an agent capable of lysing said target microorganism, preferably a phage;
providing conditions that permit the agent to lyse the microorganism if present; and
detecting the presence of non-lysed cells in the first and second parts.
2. A method for determining the presence or absence of a target microorganism in a sample comprising:
dividing the sample into at least a first part and a second part;
exposing the first part of the sample to a phage capable of infecting said target microorganism;
providing conditions that permit the phage to infect and lyse the
microorganism if present;
collecting a cell free extract from the first part and a cell free extract from the second part;
adding a microorganism susceptible to phage infection to said cell free extracts;
providing conditions that permit the phage, if present, to infect and lyse the added microorganism;
detecting the presence of non-lysed cells in the first and second parts.
3. The method of claims 1 or 2, wherein a decrease in non-lysed cells in the first part in comparison to the second part indicates the presence of the microorganism in the sample.
4. The method of any one of claims 1-3, wherein the method further comprises exposing the sample to a binding agent immobilized on a solid substrate, preferably wherein the binding agent is an antibody and preferably wherein the solid support is immunomagnetic beads, the binding agent being capable of binding the target microorganism such that the microorganism becomes associated with the solid substrate.
5. The method of any one of claims 1-4, wherein the method further comprises a selective growth step.
6. The method of claim 5, wherein the method further comprises at least one pre-enrichment growth step, preferably the selective growth step follows pre- enrichment step.
7. The method of any one of claims 1-6, wherein the method further comprises exposing both first and second parts to a reagent that labels non-lysed cells, preferably wherein the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, and a labelled binding agent capable of binding the microorganism, and detecting said reagent to determine the presence of non-lysed cells in the first and second parts, preferably wherein the reagent that labels non-lysed cells is detected using flow cytometry.
8. The method of any of claims 1-7, wherein the target microorganism is Salmonella and the phage is a Salmonella bacteriophage or a mixture of
Salmonella bacteriophages.
9. A diagnostic kit for the detection of a target microorganism in a sample comprising:
a) a phage; and b) a reagent that labels non-lysed cells.
10. The diagnostic kit of claim 9, further comprising a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism if present in the sample such that the microorganism becomes associated with the solid substrate, preferably wherein the solid support is immunomagnetic beads, preferably wherein the binding agent is an antibody.
11. The kit of any of claims 9-10, wherein the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, and a labelled binding agent capable of binding the microorganism.
12. The kit of any of claims 9-11, further comprising a microorganism susceptible to infection by said phage.
13. The kit of any of claims 9-11, further comprising a microorganism that is not susceptible to infection by said phage.
14. The kit of any of claims 9-13, wherein the target microorganism is
Salmonella and the phage is a Salmonella bacteriophage or a mixture of Salmonella bacteriophages.
15. The method of any of claims 1-8 or kit of any of claims 9-14, wherein the microorganism is a bacterium and the phage is a bacteriophage.
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