Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-06-20
2002-03-12
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S038000, C435S061000, C435S007360, C435S039000, C435S029000, C435S016000, C435S007200, C435S885000, C435S036000
Reexamination Certificate
active
06355449
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of chemistry, biology and microbiology and relates to methods and compositions for detecting the presence of vancomycin-resistant Enterococci in a sample of a possibly contaminated material.
BACKGROUND OF THE INVENTION
Enterococci are gram-positive bacteria that inhabit the gastrointestinal tract of healthy individuals. These bacteria have been identified as opportunistic pathogens for humans. Diseases caused by Enterococci include endocarditis, enterococcal bacteremia, urinary tract infections, neonatal infections, central nervous system infections (rare), intraabdominal and pelvic infections. Enterococci have emerged as one of the leading causes of nosocomial infections, responsible for 10% of all infections acquired in the hospital (Emori, T. G. and Gaynes, R. P.
Clin. Microbiol. Rev.
6:428-42, 1993).
Recent alert about Enterococci is caused not only by their increasing role in nosocomial infections, but also by their resistance to vancomycin, an antibiotic that has been used for treating infection caused by gram positive cocci. Vancomycin resistant Enterococci, emerged as the nosocomial pathogen of the 1990s, have only been discovered in the late 1980s. From 1989 through 1993, the percentage of nosocomial infections reported by the United States Center for Disease Control's National Nosocomial Infections Surveillance system that were caused by vancomycin-resistant Enterococci increased from 0.3% to 7.9% (CDC, MMWR Report 1995). Vancomycin resistant Enterococci have raised the public's anxieties and prompt intense infection control measures in hospitals around the world because no known effective therapy exists for life-threatening vancomycin-resistant enterococcal infections.
Statistics based on the United States Center for Disease Control indicated that 10% of the enterococcal infections are caused by vancomycin-resistant Enterococci with an approximate 60% of mortality rate. The World Health Organization acknowledged that vancomycin-resistant Enterococci are one of the most serious threats to human health. Recommendations for preventing the spread of vancomycin resistance have been extensively discussed in
Infectious Disease Alert
vol. 14, 185, 189, 1995; 44
MMWR,
RR-12, 1995; Edmond et al.,
Clin. Inf. Dis.
20:1126, 1995; 59 Federal Register 25758, 1994; and 16
Infection Control Hospital Epidemiology,
105, 1995.
Prompt detection and reporting of vancomycin-resistant Enterococci isolates are critical for preventing endemic spread of vancomycin-resistance Enterococci and allowing proper treatment once the right drug becomes available.
Currently, numerous vancomycin-containing selective media are used in the surveillance of vancomycin-resistant Enterococci. There is, however not a commercially available method which allows accurate, easy, and rapid detection of this important nosocomial pathogen. Recently, Landman et al.,
J. Clin. Microbiol.
(1996) 34:751-752, described the use of five selective media or identifying fecal carriage of vancomycin-resistant Enterococci.
A common procedure for detecting vancomycin resistant Enterococci by all these available methods involves adding a suspect specimen into a sterile culture medium containing all the necessary elements for bacterial growth. The media may be a liquid medium or a solid agar medium. The sample may be natural or pretreated, as by transporting the sample in a preservative medium before adding it to the selective culture medium and the medium often contain vancomycin to selective for vancomycin-resistant Enterococci. Usually, these culture media are sterilized to prevent interference from contaminating microbes, and an incubation period of from 48 to 72 hours are required for detection of vancomycin-resistant Enterococci.
One major problem for using these types of selective media is that many bacteria are intriscally resistant to vancomycin. Examples include almost of the gram negative bacteria and some gram positive bacteria (Lactobacillus spp. Pediococcus spp., and Leuconostoc spp.). Once growth is observed in these culture media, the target microbes must be isolated and confirmed through selective isolation and one or more tests specific for a variety of physiological and biochemical characteristics. Often, a number of specific colonies must be sequentially tested. In some cases, the overgrown gram negative bacteria (such as the swarming Proteus spp.) on the culture plates prevent accurate identification of specific colonies for subsequent tests. Additionally, the isolated cultures must be confirmed through antibiotic susceptibility tests for vancomycin resistance.
These methods are labor intensive, time consuming, and require highly skilled medical technologists or microbiologists to perform the tests. The above described methods usually take at least 2-3 days to complete, and are suspectible to false positives and false negatives.
The use of chromogenic or fluorogenic enzyme substrates have been widely used in microbial diagnostic methods. For example, Edberg (U.S. Pat. No. 4,925,789) described using a nutrient indicator which not only serves as a nutrient indicator, but also changes color upon being metabolized. This patent, herein incorporated by reference, provides a medium containing a nutrient indicator which, when metabolized by target bacteria, releases a moiety which imparts a color or other detectable change to the medium. The procedure takes advantage of enzyme specificity unique to particular speciies of groups of bacteria. It describes using antibiotics to select for growth of the target microorganisms and provides a specific example of liquid based assay.
Kilian et al.
Acta Path. Microbiol. Scand.
Sec. B &dgr;7 271-276 (1979) and Demare et al.,
J. Food Science
50:1736 (1985) report use of agar-based media without antibiotics. Chen and Gu, U.S. Ser. No. 08/335,149, filed Nov. 4, 1994, incorporated by reference herein, described the use of a fluorogenic nutrient indicator, 4-methylumbelifery-&bgr;-D-glucopyranoside, in a microbe-specific medium for detecting Enterococci. Each of the above described methods, however, is not suitable for detecting vancomycin-resistant Enterococci.
The above discussion is not an admission that any of the references discussed is prior art to this invention.
SUMMARY OF THE INVENTION
The present invention provides a method and media for specific detection of target microbes in a clinical sample. One of the problems in clinical sampling is that many bacteria are physiologically or biochemically similar, since these organisms reside in the same ecological system such as gastrointential tract of humans. Therefore, a simple, single enzyme reaction is often insufficient to specifically detect an organism in a medium. To achieve specific detection of target microbes, at least two enzymes should be used.
According to the present invention, a medium is provided in the method of performing a microbial diagnostic test, in which target microbes metabolize at least two nutrient indicators to yield detectable signals, and in which the presence of target microbes is indicated by the detectable characteristics yielded by two specific enzymatic reactions. The specific enzymes include &bgr;-glucosidase and pyrrolidonyl arylamidase.
Preferrably, the two or more nutrient indicators yield distinctively different detectable signals so that the presence of both or more detectable signals is distinctively detectable from the presence of only one or some of the detectable signals. In such a case, the two or more nutrient indicators can be detected at the same or about the same time. For example, one nutrient indicator gives a color in the visual range while another nutrient indicator produces fluorescence under a ultraviolet lamp.
However, in designing aqueous assay systems using two chromogenic or two fluorogenic compounds as nutrient indicators, it s often difficult or even impossible to find two nutrient indicators with different colored products, or whose signals do not interfere with each other. Obviously, two indi
Chen Chung-Ming
Edberg Stephen C.
Howrey Simon Arnold & White , LLP
Idexx Laboratories Inc.
Li Bao Qun
Salimi Ali R.
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