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Methods, compositions, and kits for the detection of bacteria in a sample

Methods, compositions, and kits for the detection of bacteria in a sample description/claims

This application is a continuation of U.S. patent application Ser. No. 11/149,979, filed Jun. 9, 2005, now pending, which application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/578,912 filed Jun. 10, 2004, which applications are incorporated herein by reference in their entireties.

1. Field of the Invention

The present invention relates to analytical methods of detecting the presence of bacteria in a sample. In particular, the invention relates to devices and methods suitable for the rapid detection of bacteria in a liquid sample, which may be used in a variety of settings, including homes, production facilities, clinics, laboratories, and the field.

2. Description of the Related Art

Bacterial contamination of water, beverages, food, pharmaceutical products, and other products ingested, used or excreted by humans or other animals is a relatively common source of infection and associated disease and is, consequently, a large concern for various food and drug manufacturers. Bacterial contamination is of particular concern to industries that produce and sell beverages, blood-related products, and pharmaceutical preparations, since these are directly introduced into the human or animal body. While the sources of bacterial contamination vary between industries, all industries attempt to minimize contamination and detect any contamination prior to the sale or use of the contaminated product.

Bacterial contamination is a continuing problem in the dairy industry (Ravanis and Lewis, Lett. Appl. Microbiol. 20:164-167, 1995). The Department of Agriculture has reported that over a billion pounds of milk are destroyed each year due to shelf-life expiration and microbial contamination (Monterubbio, Food Quality, September-October, pgs. 40-41, 2002). The occurrence of bacteria in raw milk typically comes from two sources, mastitis organisms in the udder and bacterial contamination of the teats from the environment (Reinemann et al, presented at the Annual Meeting of the National Mastitis Council, 1997). Procedures to reduce the bacterial load of raw milk have been dramatically improved in recent years; however, the problem of bacterial contamination still persists (Reinemann et al, supra).

Although standards vary from one dairy to another or from one state to another, all raw milk must be tested for its total bacterial content. The legal limit stated by the Pasteurized Milk Ordinance is 100,000 bacteria/ml for Grade A milk. Although this level of bacterial load is considered technically acceptable, most dairy processors strive for much more stringent standards. In many dairies, a standard of 10,000 bacteria/ml has been set and some set their standard at 1,000 bacteria/ml or 5,000 bacteria/ml. Every farm strives to maintain compliance with federal, state and local requirements (Reinemann et al., supra).

Raw milk is routinely tested by the standard plate count (SPC), which determines the number of colony forming units in a ml of milk following incubation at 32° C. for 48 hours. This test is almost always carried out in the dairy processor's laboratory or a contract laboratory and is rarely conducted at the farm level. Most dairies also carry out tests for coliforms, molds and total bacterial counts following incubation of the milk sample at 55° F. for 18 hours. This latter test provides information of the level of psychrotrophs in the sample (Sorhaug and Stepaniak, Trends Food Sci. Technol. 8:35-41, 1997). All of these tests are critical for determining the quality of raw milk prior to processing. Moreover, they have contributed significantly to improved milk quality, but they still suffer from the disadvantage of requiring long incubation times. Since SPCs take at least 24 hours to complete, raw milk is comingled from individual farms before the bacterial load has been established. Currently, truck drivers collect samples from multiple farms and transport them to the dairy for processing (Wetzel, Inland Northwest Dairy, Spokane, Wash., 2001, personal communication). By this time the milk is mixed and ready for processing.

A number of representatives from the milk industry have requested a rapid on-the-farm type screen that could assess the bacterial load of raw milk prior to its acceptance from the farm. This would be similar to the screening process for beta lactam antibiotics that is often conducted by the truck driver prior to receiving milk from a given farm. The dairy industry would benefit from the development of a rapid test that is inexpensive, easy to use and could be applied by farmers and truck drivers as well as dairy processors for rapidly assessing the microbial content of raw milk.

Another example of an industry faced with significant problems associated with bacterial contamination is the blood product industry. Bacterial contamination can occur in a variety of blood-based products used to treat humans, including, e.g., whole blood, red blood cells and platelets.

Bacterial contamination of platelets is of particular concern, since these organisms survive and readily multiply at temperatures of 20 to 24 degrees C., the storage temperature of platelets. Although the incidence of platelet contamination is greater from gram-positive organisms, fatalities due to platelet contamination tend to be equally divided between organisms that are gram-positive and gram-negative. The organisms most commonly implicated in fatalities, in descending order, are Staphylococcus aureus, Klebsiella pneumoniae, Serratia marcescens, and Staphylococcus epidermidis. Other isolated organisms include Salmonella sp., Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus. Skin contaminants, such as Staphylococcus epidermidis and Bacillus sp. are the organisms most implicated in platelet bacterial contamination. The seriousness of this problem is exacerbated by the fact that Staphylococcus aureus is the leading cause of nosocomial (hospital acquired) infections and is increasingly becoming resistant to conventional antibiotic therapies.

Bacterial contamination of transfusion products have been known as a potential source of harm since the beginning of transfusion history, but so far only a handful of countries actually test platelet products for contamination. In recent years, the focus has been directed towards viruses such as HIV and HCV, and new methods for detection have reduced the risk greatly. However, it is realized that the frequency of bacterial contamination of blood platelets and the incidence of illness and fatalities caused by bacterial contamination, greatly exceed that of viruses.

Detection of bacteria in platelets is difficult, mainly due to the very low initial inoculum present in the product. In addition, platelets may be contaminated with a range of bacteria that will grow at different rates. This makes sampling a major challenge to developers and users of test systems, and may cause the presence of bacteria in a product to be missed due to sampling error. Another challenge is the short shelf life of platelets (5-7 days). It is therefore very important to have a rapid and reliable method. Current methods may take days before a positive result is obtained, leaving very little shelf life for the products.

Methods, such as visual inspection or platelet swirling, although easy to implement, lack sensitivity resulting in an unacceptably high rate of false positives and the unnecessary destruction of viable and valuable platelets.

Metabolic methods, such as multi-agent indicator strips, which measure bacterial growth by decreases in glucose and pH levels, offer rapidity, but lack sensitivity, since several bacterial species have metabolic characteristics not responsive to these markers.

Traditional culture techniques require a lengthy incubation period of several days. False positives and false negatives may occur due to a variety of circumstances including the low number of bacteria present and incubation temperatures for a given organism. Automated bacterial culture tests, which are used in some European countries, have many of the same drawbacks found in the standard culturing techniques. They lack specificity for some types of platelet contaminating organisms and are unable to detect the presence of slow-growing bacteria.

Accordingly, there is a need in the art for methods and compositions for the rapid and sensitive detection of bacteria in liquid samples, including beverages and pharmaceutical preparations, such as milk, water, and blood products.

In a first embodiment, the invention includes a device for the detection of bacteria, comprising a carrier to which a liquid sample suspected of containing bacteria can be applied, a binding agent that specifically binds bacteria, and a detection means. The device may further comprise a second binding agent that specifically binds bacteria. In one embodiment, a binding agent is immobilized in a first region of the carrier. In one embodiment, a binding agent binds peptidoglycan or a component thereof. In certain embodiments, a binding agent is labeled, while in other embodiments, it is not labeled.

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