Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1998-07-27
1999-11-16
Fonda, Kathleen K.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 5, 435236, 4352531, 435259, 435864, 435866, C12Q 106, C12Q 170, C12N 106, C12N 704
Patent
active
059855967
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method, notably to a method for diagnosing the presence of a bacterium in a sample.
BACKGROUND TO THE INVENTION
It is common practice to propagate bacteria from a sample of a bodily fluid, such as sputum, on a suitable culture medium so as to be able to detect the colonies of bacteria on the culture medium either visually or using a test reagent or assay. Such a technique is applicable over a wide range of bacteria and has become widely accepted as a standard bacteria detection and diagnostic procedure. However, problems arise where the bacterium is a slow growing type where it may take several days or even weeks before adequate growth of the bacteria has taken place to be detectable. This is particularly the case with some Mycobacterium or Legionella species, where it can take up to four weeks or more to propagate detectable colonies of the bacteria. This enforces an unacceptable delay in confirming infection with the bacteria in patients and it would be desirable to provide a more rapid assessment of the presence and identity of the bacteria.
It has been proposed in PCT Application No WO92/02633 to prepare a sample of a viable bacterium and to infect that with a strain of a virus which is specific to that bacterium, such bacteria-infecting virus being denoted as bacteriophage or, more commonly, phage. Some of the phage particles infect the bacteria and replicate within the bacterial cell. Those phage particles which are not absorbed by the bacteria remain with the mother liquor or matrix of the carrier of the bacteria, typically a culture medium. If the infected sample is then treated, for example with heat and/or an acid to kill, or is washed with a surfactant to remove, the exposed, exogenous, phase particles, the exogenous phage particles are inactivated or removed. The infecting, endogenous, phage particles are not inactivated, since they are protected within the bacterial cell. The treated infected bacteria can then be cultured in the presence of further un-infected bacteria which are permissive to, that is can be infected by, the phage particles. The phage particles within the infected bacteria replicate causing the cell wall to rupture or lyse and release its load of replicated phage particles. This first generation of progeny phage particles infects other bacterial cells, causing successive cycles of infection replication and lysis. This gives an exponential increase in the number of phage particles in the culture medium. Either the reduced growth of the bacterial cells or the large number of replicated phage particles can be measured or observed, even where the initial number of infected bacterial cells was small, thus enhancing the sensitivity of the detection of the presence of the initial bacterial cells in the sample being assessed.
This modification enhances the sensitivity of detection of bacterial cells and has been used successfully in the detection of fast growing bacteria such as Salmonella spp. However, with slow growing bacteria such as Mycobacteria spp, we have found that the time taken for this procedure to give a detectable population of phage particles remains substantially the same as with other techniques and the procedure does not solve the time problem with such bacteria. The need still exists for a procedure which achieves rapid and accurate detection of slow growing bacteria.
We have devised a method by which slow growing bacteria can be rapidly detected, thus overcoming the problems in having to wait days or even weeks for such detection. The method can also be used to determine the sensitivity of given bacteria to antibiotic and other drugs and to determine whether a given antibiotic or drug is active against given bacteria or whether a given virucidal composition is effective against a given virus. Since the method can be performed using simple apparatus and by unskilled persons, the method readily lends itself to operation in third world countries where skilled microbiologists and complex apparatus may not be available.
SUMMARY OF
REFERENCES:
patent: 5498525 (1996-03-01), Rees et al.
Biotec Laboratories Limited
Fonda Kathleen K.
Moran Marjorie A.
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