Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-10-21
2001-03-20
Minnifield, Nita (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007350, C435S008000, C435S007940, C435S007320, C435S007900, C435S975000, C435S870000, C435S007950, C435S040510, C530S387100, C530S388400, C530S389500
Reexamination Certificate
active
06203996
ABSTRACT:
The present invention relates to a means of rapid detection of bacteria from a liquid culture or slurry. The liquid culture may be a true liquid such as milk but it may also be produced by suspending a solid in a liquid or mincing or macerating the solid therein.
The attachment of bacteria to solid surfaces is a well known phenomenon. The ease with which microorganisms accumulate at surfaces is the cause of numerous economic and biological problems. Microorganisms will readily colonize man-made structures immersed in aqueous environments which can lead to corrosion and fouling (Herald and Zottola, 1988). Many diseases of animal and plants result from the growth of pathogenic microorganisms on host epithelial surfaces (Walker and Nagy, 1980; Dazzo, 1980).
The attachment of bacteria to food surfaces including meat contributes to food spoilage and the risk of food poisoning (Selgas et al., 1993, Firstenberg-Eden, 1981), for example,
Listeria monocytogenes
is an important food borne pathogen which may contaminate meat, cheese and other foodstuffs. A number of authors have reported the attachment of
L. monocytogenes
to solid surfaces including glass, stainless steel, polypropylene and rubber surfaces (Dickson and Daniels, 1991, Mafu et al. 1990, Fletcher and Loeb, 1979).
Jeong-Weon et al. (1993) described the adhesion of
Salmonella typhimurium,
another important food pathogen, to turkey skin. These authors showed that the attachment of the pathogen was much higher to skin surfaces on which the collagen fibers were exposed. This suggests that there is a specific receptor in the collagen fiber which binds to Salmonella. This view was corroborated by Walls et al., (1993) who used sausage casing as a model for the attachment of Salmonella to meat. The attachment of Salmonella to collagen fibers can be used as a model for the attachment of a number of important pathogens to meat.
Bacterial attachment to solid surfaces is believed to be influenced by cell surface charge (Fletcher & Loeb, 1979),hydrophobicity (van Loosdrecht et al., 1987) and by the presence of particular surface structures such as flagella, fimbriae, and extracellular polysaccharides (Fletcher & Floodgate, 1973). Abolosom et al., (1983) reported that attachment was greatest for hydrophobic organisms. Of five organisms examined by these authors,
L. monocytogenes
was the most hydrophobic and showed the greatest amount of attachment.
It is possible that an increased concentration of a particular bacterial species in a liquid medium leads to an increase in the number of collisions with the surface (Fletcher, 1979). Chung et al. (1989) reported that in the simultaneous presence of
L. monocytogenes
and
Pseudomonas aeruginosa
no significant competitive attachment between the two species occurred. This is relevant because previous studies have shown that the pseudomonads form part of the meat microflora growing during incubation of both selective and non selective enrichment broths (Duffy et al., 1994). Farber and Idziak (1984) also reported that, in general, the attachment of one organism to meat was unaffected by the presence of another organism.
Many rapid methods for the detection of bacteria employ specialised kit systems which are very expensive (Listeria-tek, Gene-trak). Techniques such as Gene-trak are labour intensive while flow cell cytometry techniques require operators to have specialised training (Donnelly and Baigent, 1986). The electrical based methods such as conductance and impedance are currently the simplest to carry out and have been automated.
EP-A-0 429 794 A2 discloses an assay method for detecting Listeria which is an immunoassay characterised by the combined use of antibodies specific for two separate cell wall components, namely peptidoglycan and teichoic acids, the antibodies being immobilised on a solid support.
EP-A-0 498 920 A2 also relates to an assay method for detecting the presence of bacteria or other culturable organisms. It involves an immunoassay method to detect the presence of viable bacteria strains in foods and other potentially contaminated samples using an assay characterised by
(1) capture of specific bacteria cells with specific antibodies immobilised on a solid support;
(2) incubation of the captured cells to form bacteria colonies;
(3) imprint of the colonies to a colony lift membrane; and
(4) immunochemical detection and species identification of the colonies on the colony lift membrane.
EP-A-0 281 477 A1 describes a method in which spores are detected by coupling the spores with a labelled monoclonal anti-spore antibody, depositing the resultant antigen—antibody complex on a gel containing a substrate for the label and a detector syste to show deposited complex. U.S. Pat. No. 3,989,591 describes a method of isolating and/or separating bacteria and tissue cells for testing or rapid automatic screening using an expandable membrane on which the tissue cell or bacteria sample is placed and subsequently expanded to cause the sample to be separated. EP-A-0 511 110 A2 describes a method of separating bacteria comprising passing bacteria-containing fluid through a ceramic membrane on a ceramic support in order to sterilise the liquid by filtration.
It is an object of the present invention to provide a method for the isolation and detection of bacteria from a liquid culture, which is simple to perform, rapid, inexpensive, non-labour intensive and sensitive. In particular, the invention seeks to provide a method for the detection of microbial pathogens in foodstuffs. The invention also seeks to provide a method of detection of microorganisms for use as a clinical or medical diagnostic test.
The present invention provides a method of rapid detection of bacteria in a liquid culture suspected to contain bacteria wherein a membrane mounted on a support is immersed in a liquid culture for a time sufficient to allow bacteria to adhere to the membrane, the membrane is removed from the liquid culture and the number of bacteria adhering to the membrane is counted.
The membrane may be an inanimate membrane such as a polycarbonate membrane; a membrane based on a cellulose derivative such as an acetate, nitrate or ester derivative; a polyvinyl chloride membrane; a polyamide membrane; a nylon membrane or an inorganic membrane such as a silver or aluminium membrane. An ion exchange membrane may be used to give a selective separation of the organisms.
Alternatively the membrane may be a biological membrane such as animal skin, animal intestinal membrane or other animal internal membrane, or a sausage casing membrane or a collagen membrane. Biological membranes may be advantageous for more selective isolation of microorganisms.
The solid support on which the membrane is mounted may be a glass microscope slide, plastics slide, wire or other suitable frame. The solid support should be sterilisable. Any support to which a membrane can be temporarily be attached is suitable for use.
The membrane may be immersed in the liquid culture for a least 10 minutes, and preferably at least 15 minutes, at between 25 and 30° C.
Preferably the method also comprises the step of coating the membrane with a labelled anti-bacterial antibody following removal of the membrane from the liquid culture. The antibody is most suitably a monoclonal antibody.
The anti-bacterial antibody may be an anti-Listeria antibody, or an anti-Yersinia antibody.
The membrane may be washed between removal from the liquid culture and coating with the antibody.
The label may be selected from fluorescein isothiocyanate, tetramethylrhodamine, horseradish peroxidase, alkaline phosphatase, glucose oxidase or any other label known in the art.
If a fluorescent label is employed, the membrane may be examined under an ultra-violet light microscope and a count of fluorescing labelled bacterial cells made, preferably, in a plurality of fields. Alternatively a laser system may be used to scan the membrane and count labelled cells.
The liquid culture may be prepared by immersion of a solid or semi-solid substance in a liquid such as an enrichment br
Duffy Geraldine
Sheridan James
Minnifield Nita
Morrison & Foerster / LLP
Teagasc, The Agriculture and Food Development Authority
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