Method to detect bacteria

Details Method to detect bacteria Method to detect bacteria

2000-06-15

2002-10-08

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S036000, C435S038000, C435S005000

Reexamination Certificate

active

06461833

ABSTRACT:

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 WO92/02533 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 bacteria 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, phage 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 phase 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 Mycobacterium spp, we nave 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 THE INVENTION
Accordingly, from one aspect the present invention provides a method for detecting the presence of first bacterium in a sample, which method comprises the steps of:
a. infecting a viable sample of the first bacterium cells with a phage which is specific to that bacterium whereby at least some of the bacterium cells each absorb one or more of the phage particles;
b. inactivating or removing the phage particles which have not been absorbed by the bacterium cells;
c. cultivating the infected first bacterium cells in a medium containing at least one other second bacterium which has a reproduction rate greater than the reproduction rate of the said first bacterium whereby at least some of the said second bacterium cells become infected by phage particles released upon the lysis of said infected first bacterium cells, each of said infected bacterium cells acting as a source for further infection of a subsequent generation of the infection cycle; and
d. monitoring either the population of replicated phage particles and/or the reduced growth of the said second bacterium in stepc to determine the presence of said first bacterium cells in the sample under test.
Preferably, the cultivation and infection of the second bacterium is carried out to or near the steady state at which further bacterial cell growth and lysis substantially ceases. At this state, a substantially steady population of dead bacteria cells due to the activity of the phage particles is present in the cultivation medium. The areas of the cultivation medium in which phage replication has taken place can be detected visually as clear spots in the surrounding cultivation medium which is more cloudy, opaque or otherwise visually different due to the growth of viable bacteria. These areas of reduced bacterial growth due to phage activity are commonly termed plaques. Since the second bacterium reproduces at a greater rate than the first bacterium, the time taken for the initial infected first bacterium cells to produce detectable plaques is greatly reduced. This acceleration in the development of plaques does not occur when the same bacterium is used for the initial phage infection and for the subsequent cultivation steps.
Alternatively, the presence of replicated phage particles in the cultivation medium at the end of stepc can be assessed using conventional phage assay techniques. This can be done in addition to or as an alternative to the determination of the existence of plaques as described above.
The method of the invention can thus be applied to the detection of slow crowing bacteria in a sample, for example from a patient suspected of being infected with
Mycobacterium tuberculosis
or Legionnaires disease, where a conventional assessment would takes days or weeks to give any observable indication that such bacteria were present in the initial sample. In such a case, a proportion or the initial sample is infected with a phase specific to the expected bacterium and the phage particles are carried through the in-activation step b because they are protected by the bacterial cells which they have infected. These protected phage particles cause the development of phage or plaques in the environs of each infected first bacterium cell in the cultivation of the infected cells—stepc. If none of the suspected bacteria were present in the sample, substantially no phage or plaques would be formed in stepc and the cultivation medium would present a substantially uniform appearance.
The invention may also be carried cut using a known bacterium which has been treated with an antibiotic or other drug under test to determine whether the antibiotic or drug has any bactericidal effect. In this case, the population of phage or plaques at the end of stepc indicates the efficacy or otherwise of the antibiotic or drug in killing the initial bacterium. In a conventional test method, the antibiotic or drug would be applied to the bacterium and the number of dead cells would be a direct indication of the efficacy of the antibiotic o

Affiliated with

Also associated with

Rating

Method to detect bacteria does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method to detect bacteria, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method to detect bacteria will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2923862