Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-07-19
2002-06-25
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S030000, C435S288300
Reexamination Certificate
active
06410256
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and apparatus for the incubation and analysis of biofilms, and analysis of sensitivity of biofilms to antimicrobial reagents.
BACKGROUND OF THE INVENTION
Extensive study into the growth properties of bacteria in recent years has shown that they form complex layers that adhere to surfaces. These complex forms of bacteria are known as biofilms, or sessile bacteria. Biofilms may cause problems in a variety of areas including the bodies of humans and animals, food processing, health care facilities, metal-working shops, dairy farms and other industries.
It is now widely known that bacteria in the form of biofilms are more resistant to antimicrobial reagents than planktonic bacteria. Yet testing for the presence of bacteria and the testing of the efficacy of antibiotics against bacteria has traditionally involved testing for planktonic bacteria. Thus, bacterial inhibitory concentration of the antimicrobial reagent may be underestimated, with the result that the wrong antimicrobial reagent or wrong amount of antimicrobial reagent may be used for the treatment of the bacteria.
One type of device for monitoring biofilm buildup is described in the Canadian Journal of Microbiology (1981), volume 27, pages 910 to 917, in which McCoy et al describe the use of a so-called Robbins device which comprises a tube through which water in a recycling circuit can flow. The tube has a plurality of ports in its walls, each being provided with a stud having a biofoulable surface and being capable of being retained in the port in fixed relationship with respect to the tube so that the biofoulable surface forms part of the internal surface of the tube. The studs may be removed from the ports after a desired time interval and the test surfaces by microscopy of the surfaces analyzed for the growth of microorganisms or by removal of the microorganisms from the surfaces and subsequent estimation of the degree of growth. The number of microorganisms can be estimated for instance by physical or chemical means, e.g. by detection of bacterial ATP or by further culturing the microorganisms and analyzing the products.
In another device described in U.S. Pat. No. 5,349,874, biofilm growth in a water carrying conduit is determined by providing plural removable studs in the conduit or in a second conduit parallel to the first. The studs may be removed for analysis of biofilm on the studs. Such devices as the Robbins device, or others using removable studs in a single conduit, result in rather lengthy processing times, and do not provide rapid response times for the testing of several different antimicrobial reagents.
In a still further device, described in “Simple Method for Measuring the Antibiotic Concentration Required to Kill Adherent Bacteria”, Miyake et al, Chemotherapy 1992; 38, 286-290,
staphylococcus aureus
cells adhered to the bottom of a 96 well plastic tissue culture plate were treated with serially diluted antibiotic solutions, and viability of the cells was judged by their growth after a further 24 hours incubation. This method has the disadvantage of inconsistent colonization of sessile bacteria and settling of planktonic bacteria.
The device described in this patent document allows for an efficient and automated biofilm killing assay that has particular use with the 96 well platform common to many diagnostic assay systems.
SUMMARY OF THE INVENTION
There is therefore provided in accordance with one aspect of the invention, a method of growing a biofilm, in which biofilm forming organisms are incubated to form a biofilm on plural biofilm adherent sites by providing a flow of liquid growth medium across the plural biofilm adherent sites, the direction of the flow of liquid being repeatedly changed.
In a further aspect of the invention, biofilm forming organisms are incubated to form a biofilm on plural biofilm adherent sites arranged in plural rows, with plural biofilm adherent sites in each row, while providing a flow of liquid growth medium across the plural biofilm adherent sites.
In a further aspect of the invention, an assay is made of a characteristic of the resulting biofilms.
In a further aspect of the invention, the characteristic of the biofilm is the sensitivity of the biofilm to antimicrobial reagent and the method further includes, before assaying the biofilm, treating the biofilm adherent sites with antimicrobial reagent.
In a further aspect of the invention, there is also included the step of, after treating the biofilm adherent sites with antimicrobial reagent, dislodging the biofilm from the biofilm adherent sites and further incubating the biofilm. Dislodging the biofilm from the biofilm adherent sites may include dislodging the biofilm from each biofilm adherent site into a separate well of a microtiter plate.
When the biofilm adherent sites are formed in rows, treating the biofilm adherent sites with an antimicrobial reagent may include treating each row of biofilm adherent sites with a different antimicrobial reagent, and treating each of the biofilm adherent sites in a row with a different concentration of antimicrobial reagent.
In a further aspect of the method of the invention, different biofilm adherent sites are treated with different antimicrobial reagents, such as different combinations of antimicrobial reagents as might be used in testing the efficacy of various modifications of a single antimicrobial reagent.
Preferably, the flow direction of the liquid growth medium is repeatedly reversed. In this aspect of the invention, the liquid growth medium may flow in channels of a vessel, and the direction of flow of the liquid growth medium is reversed by rocking of the vessel.
In a further aspect of the invention, the biofilm adherent sites are projections from a lid and incubating the biofilm includes suspending the projections in liquid growth medium in the channels while rocking the vessel so as to provide shear forces on the biofilm during growth of the biofilm.
There is further provided, in accordance with an aspect of the invention, apparatus for analyzing biofilms, the apparatus comprising a vessel including at least one channel for flow of liquid growth medium. Plural biofilm adherent sites are arranged in at least one row and have a support for supporting the plural biofilm adherent sites within the channel.
In accordance with a further aspect of the apparatus according to the invention, there is provided means to flow liquid growth medium along each channel, preferably in different directions, across the plural biofilm adherent sites.
In accordance with a still further aspect of the apparatus of the invention, there is provided means to avoid contamination of the inside of the vessel.
Preferably, the plural biofilm adherent sites are formed in plural rows, with plural sites in each row, and the vessel includes plural channels, with one channel for each row of plural biofilm adherent sites.
In a further aspect of the invention, the support for the plural biofilm adherent sites forms a lid for the vessel.
In a still further aspect of the invention, the means to flow liquid growth medium across the plural biofilm adherent sites is a tilt table.
These and other aspects of the invention will be made apparent in the description and claims that follow.
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Gjaltema, A., et al., “Heterogeneity of Biofilms in Rotating Annular Reactors: Occurrence, Structure, and Consequences”,Biotech. Bioeng., 44:194-204 (1994).
Zimmerli, W., et al., “Microbiological tests to predict treatment outcome in experimental device-related infections due toStaphylococcus aureus”,J. Antimicrob. Chemo., 33:959
Buret Andre Gerald
Ceri Howard
Morck Douglas Walter
Olson Merle Edwin
Read Ronald Rae
Burns Doane Swecker & Mathis L.L.P.
Gitomer Ralph
University Technologies International Inc.
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