Rapid identification of microorganisms

Details Rapid identification of microorganisms Rapid identification of microorganisms

2000-09-05

2002-08-06

Tate, Christopher R. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S005000, C435S007370, C435S029000, C435S030000, C435S034000, C435S173100

Reexamination Certificate

active

06428976

ABSTRACT:

BACKGROUND OF THE INVENTION
Escherichia coli
(“
E. coil
”) O157:H7, first isolated in 1975 from a patient with grossly bloody diarrhea, is now recognized as an important foodborne pathogen (Ministry of Health and Welfare, Information on the Detection of Pathogenic Microorganisms, 1996; Meng et al., Trends Food Sci. Tech. 5:179-185, 1994; Padhye et al., J. Food Prot. 55:555-565, 1992; Riley et al., N. Engl. J. Med. 308:681-685, 1983). In adults, the illness is usually self-limited. However, the more serious hemolytic uremia syndrome (“HUS”) affects some of the infected patients, especially children and the elderly. The mortality rate of HUS is 3 to 10%. Most outbreaks have been associated with consumption of undercooked ground beef or raw milk. Cattle has been identified as an important reservoir of
E. coli
O157:H7. Person-to-person transmission has also been identified in some day-care center and nursing home outbreaks (Centers for Disease Control and Prevention, Morbid. Mortal. Weekly Rep. 42:253-257, 1993; Lior, Dairy Food and Environ. Sanitation 14:378-382, 1994; Padhye et al., J. Food Prot. 55:555-565, 1992).
Several methods have been developed for rapid detection and identification of
E. coli
O157:H7. Most of these methods are immunoassays for detecting the
E. coli
157
somatic antigen (Huang et al., J. Food Prot. 59:170-174, 1996; Meng et al., Sci. Tech. 5:179-185, 1994). Confirmation of a positive result by either biochemical or serological tests, such as Vero cell assay or a test for the presence of H7 antigen, are required.
DNA probes and polymerase chain reaction have also been used to detect
E. coli
O157:H7 (Meng et al., Sci. Tech. 5:179-185, 1994). Although DNA-based methods are sensitive, there are several major drawbacks. First, they detect the presence of nucleic acid of the target bacteria rather than the viable bacteria themselves. Second, most of the DNA-based methods are designed to detect the genes that encode verotoxin or virulence-associated marker of verotoxin- producing
E. coli
, and are not specific for
E. coli
O157:H7. In addition, DNA-based methods are cumbersome and expensive.
A coliphage named AR1 has been found to infect
E. coli
O157:H7 with high specificity (Ronner et al., Journal of Food Protection 54: 944-947, 1990).
SUMMARY OF THE INVENTION
The invention features a method of determining whether a test microorganism is a known microorganism such as a bacterium (e.g., an enterobacterium such as
Escherichia coli
), or yeast. The method includes the steps of: (i) providing a first culture that contains the test microorganism and an agent (e.g., a bacteriophage) that specifically affects the growth rate of the known microorganism; (ii) measuring a growth rate-related value of the first culture; and (iii) comparing the value of the first culture with a corresponding value of a second culture, the second culture being identical to the first culture except that the second culture is free of the agent, or contains the known microorganism and is free of the agent; wherein a difference in the two values is an indication that the test microorganism is the known microorganism. Examples of growth-rate-affecting agents include, but are not limited to, AR1 phage for
E. coli
O157:H7, P22 phage for
Salmonella typhimurium
(Griffiths, J. Dairy Sci., 76:3118-3125, 1993), and A511 phage for Listeria (Stewart et al., ASM News, 62:297-301, 1996). By “Specifically” is meant that the agent affects the growth of mainly one microorganism. Of course, an agent that has cross-activity to a very limited number (e.g., no more than 3) of other microorganisms may also be used, if additional discerning criteria are available.
Also featured in the invention is a method of determining whether a test microorganism is
E. coli
O157:H7.
One embodiment of the method includes the following steps: (1) determining whether the test microorganism is
E. coli
; (2) growing the test microorganism in a medium containing sorbitol; (3) determining whether the test microorganism ferments sorbitol; (4) providing a first culture that contains the test microorganism and AR1 phage; (5) measuring a growth rate-related value of the first culture; and (6) comparing that value of the first culture with a corresponding value of a second (i.e., control) culture that is identical to the first culture except that (a) it is free of AR1 phage, or (b) it is free of AR1 phage and contains any
E. coli
strain instead of the test microorganism. The test microorganism is indicated as
E. coli
O157:H7 if (i) it is
E. coli
, (ii) it is incapable of fermenting sorbitol, and (iii) there is a significant (e.g., at least two-fold) difference in the growth rate-related value between the two cultures.
In another embodiment of the method of this invention, the test microorganism is already known to be
E. coli
. To determine if this
E. coli
strain is O157:H7, one can (1) grow the test strain in a sorbitol-containing medium; (2) determine whether the test strain ferments sorbitol; (3) provide a first culture that contains the test strain and AR1 phage; (4) measuring a growth rate-related value of the first culture; and (5) compare that value of the first culture with a corresponding value of a second culture which is identical to the first culture except that (a) it is free of AR1 phage, or (b) it contains any other
E. coli
strain and is free of AR1 phage. The test strain will be identified as
E. coli
O157:H7 if (i) it is incapable of fermenting sorbitol; and (ii) there is a significant (e.g., at least two-told) difference in the growth rate-related value between the two cultures.
Yet another embodiment of the method is to determine whether a test microorganism incapable of fermenting sorbitol is
E. coli
O157:H7. This method includes the following steps: (1) determining whether the test microorganism is indicated as
E. coli
; (2) providing a first culture that contains the test microorganism and AR1 phage; (3) measuring a growth rate-related value of said first culture; and (4) comparing that value of the first culture with a corresponding value of a second culture that is identical to the first culture except that (a) it is free of AR1 phage, or (2) it is free of AR1 phage and contains any
E. coli
strain. The test microorganism is
E. coli
O157:H7 if (i) it is
E. coli
; and (ii) there is a significant (e.g., at least 10-fold, or even 20-fold) difference in the growth rate-related value between the two cultures.
If the test microorganism is already known to be susceptible to AR1 infection, one can conduct the following steps to determine if the microorganism is
E. coli
O157:H7: (1) determining whether the test microorganism is
E. coli
; (2) growing the test microorganism in a culture medium containing sorbitol; and (3) determining whether the test microorganism ferments sorbitol. The test microorganism is
E. coli
O157:H7 if it is
E. coli
and is incapable of fermenting sorbitol.
In all of the above-described methods, a growth rate-related value includes, but is not limited to, (i) a value of an electrical parameter such as conductance, resistance, or any other proper parameter; (ii) a value derived from values of an electrical parameter, e.g., a time point at each an accelerating change of an electrical parameter occurs (if the electrical parameter is conductance, the time point is herein termed “detection time”; (iii) a value of an optical parameter such as optical density; (iv) a value derived from values of an optical parameter; and (v) certain biochemical indexes that reflect growth of a microorganism. The methods based on use of an electrical or optical parameter allows automated screening of a large number of samples.
Other features and advantages of the present invention will be apparent from the following drawings and description, and also from the appending claims.


REFERENCES:
patent: 4160205 (1979-07-01), Hobbs et al.
patent: 4264728 (1981-04-01), Wilkins
patent: 4945060 (1990-07-01), Turner et al.
patent: 5336600 (1994-08-01), Monget
Okrend et al. J. Food Protect. 1990.

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