Bacteriophage assay

Details Bacteriophage assay Bacteriophage assay

2001-01-12

2003-04-29

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S005000, C435S007100, C435S030000, C424S204100, C424S234100, C424S093300, C424S235100

Reexamination Certificate

active

06555331

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is concerned with the identification of bacteria, both by species and by sub-type, and to a new method of bacterial identification which relies upon bacteriophage specificity.
The control and epidemiology of bacterial outbreaks is becoming increasingly important and much effort is currently expended in identification of bacteria by species and sub-type. With the apparent continued rise in antibiotic resistant bacterial strains the need for careful and accurate identification of bacteria is becoming ever more critical.
Strain typing has been defined as “A pre-requisite to studying the epidemiology of bacterial pathogens and, ultimately, the development of control strategies” (see Smith et al, (1995) AEM 61:4263).
Typically identification of bacterial species and sub-types involves methods such as classifying bacteria according to their ability to grow using selected carbon sources, the specificity of bacteriophages for particular bacteria, or involves genetic analysis of the bacterial genome and comparison thereof to known genomic sequences (for example using techniques such as RFLP, RAPD, ERIC, PCR-RFLP or the like). For example strain differentiation in
Erwinia carotovora
subspecies
atroseptica
(hereinafter referred to as “Eca”) is usually performed by serology, phage typing, carbon source utilization, genetic analysis or a combination of such techniques. All of the currently used methods are relatively time-consuming, causing delay in the positive identification of a sample. Minimisation of any delay may be vital for successfully controlling the spread of bacterial infections in the population generally or in selecting a suitable treatment regime for a patient.
For example, strain typing of bacteria may be carried out using bacteriophages (hereinafter referred to simply as “phages”). A phage is any virus whose host is a bacterium. Most bacteria can be infected by phages, which are a highly diverse group of viruses. A given phage can only infect one or a few strains or species of bacteria and this limitation of phage infectivity forms the basis of strain typing using phages. The outcome of phage infection depends upon the phage and its host cell, but can be classified into two main groups as follows:
Virulent phages: induce lysis of the host cell.
Temperate phages: can establish a stable non-lytic relationship with the host cell.
Conventionally, strain typing of an unknown strain of bacteria via phage infection involves plating out a single colony of bacteria obtained from a test sample onto an agar dish and, once a bacterial lawn is established, introducing inocula of a specific phage at discrete points. The inoculated plate is then incubated again before being examined by eye and the extent of degradation or lysis of the bacteria at the points where phage has been introduced is graded by the technician. The effect of various phages on the test bacteria are analysed.
The grading used to establish the extent of phage action on a bacterial colony will vary from complete lysis (and thus death) of the bacteria (due to successful replication of the phage) through to no effect noticeable to the eye (when the phage is unable to interact with the bacteria of the sample). Various grades between these two extremes also exist and to a large extent the accuracy of the test results depends upon the skill, experience and perception of the person reading the results and performing the grading procedure. Unfortunately, the subjective nature of the grading system means that ultimately the phage typing system lacks accuracy.
A typical analysis of
E. coli
O157:H7 by conventional phage typing methods is reported by Khakhira et al., in Epidermiol. Infect. (1990) 105:511-520, see especially Table 1 of this reference in this analysis there was visual assessment of 62 phage types and an attempt to assign a positive value to each result. The complexity of conventional bacterial strain typing by phage interaction is clear from the typical analysis results depicted in Table 1.
SUMMARY OF THE INVENTION
We have now found that the highly specific interaction between phages and bacteria can be used in a much more effective assay in which the results of the phage/bacteria interaction is determined through measurement of bacterial growth, rather than bacterial death. This novel approach to phage typing enables conventional techniques for observing bacterial populations, such as determining the optical density of a sample, to be successfully employed. Consequently the results do not rely on a subjective analysis but on a direct and reproducible reading of a characteristic of the test sample.
In the assay of the invention a predetermined amount of phage is combined with an isolate of bacteria, the mixture being located in a suitable container. The mixture of phage and bacteria is conveniently held in a liquid or semi-liquid medium facilitating interaction of the two species. Conveniently the phage is located in the container and the bacteria added to the phage. However the assay is not limited to this approach and also encompasses, for example, the bacteria being located in the container and the phage added thereto.
In one aspect the present invention provides an assay to identify bacteria present in a sample, said assay comprising the following steps:
(a) isolating a single colony of said bacteria;
(b) combining said isolated bacteria with a selected bacteriophage in a container, the combination of bacteria and phage being incubated in a medium containing the nutrients required for bacterial growth and which enables phage/bacteria interaction; and
(c) determining the extent of bacterial growth.
Where the phage interacts with the bacteria of the test sample, the phage will infect the bacteria and, depending upon the virulence of the phage, will either cause death of the bacteria or will slow bacterial reproduction. If the phage is unable to infect the bacteria, bacterial growth will be unaffected. Thus, determination of the extent of bacterial growth following incubation with the phage is a direct correlation of the interaction of the phage and bacteria. Since the phage will interact only with specific bacteria, the extent of bacterial growth in the assay provides information on the species or sub-type of the bacteria.
Thus the assay of the present invention will have utility as an in vitro method of diagnosis for bacterially based infections or diseases in plants, animals and humans. Additionally the assay has utility as a means of monitoring food or medicines etc. for bacterial contamination.
Suitable phages are commercially available from culture collections and conventional phage typing systems. In addition phages specific for particular bacteria can be engineered using routine techniques in the laboratory due to the phages' ability to rapidly mutate producing host range mutants. Alternatively suitable phages can be isolated from the natural habitat of the bacteria in question and again standard techniques and methodologies are well known and within the ability of the skilled technician.
One important feature of the present assay is to retain the phage of interest in a non-replicative state to avoid mutation of the phage prior to the assay. Similar requirements are imposed on the conventional method of phage typing and have not been found to be unduly onerous since if no bacteria are present, the phage will be unable to replicate or mutate.
Suitable media for incubation of the phage/bacteria combination include those conventionally used for growing bacterial cultures, for example nutrient broth (NB) or Luria Bertani (LB) broth. Conveniently the medium is liquid at the temperature of incubation since this assists the mixture of phage with the bacteria. It is essential that the medium chosen is compatible with the method used to determine the extent of bacterial growth. Thus if the optical density of a sample is used as a measure of bacterial growth, the medium chosen must enable the penetration of light through the sample. Liquid media are gener

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