Comparative phenotype analysis

Details Comparative phenotype analysis Comparative phenotype analysis

2002-08-23

2004-04-27

Tate, Christopher R. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S029000, C435S032000, C435S004000, C435S007310, C435S007320

Reexamination Certificate

active

06727076

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to growing and testing any cell type in a multitest format which utilizes a gel forming matrix for the rapid screening of clinical and environmental cultures. The present invention is suited for the characterization of commonly encountered microorganisms (e.g.,
E. coli, S. aureus,
etc.), as well as commercially and industrially important organisms from various and diverse environments (e.g., the present invention is particularly suited for the growth and characterization of the actinomycetes and fungi). The present invention is also particularly suited for analysis of phenotypic differences between strains of organisms, including cultures that have been designated as the same genus and species. In addition, the present invention provides methods and compositions for the phenotypic analysis and comparison of eukaryotic (e.g., fungal and mammalian), as well as prokaryotic (e.g., eubacterial and archaebacterial) cells.
BACKGROUND OF THE INVENTION
Ever since the golden age of microbiology in the era of Koch and Pasteur, methods for identification of microorganisms have been investigated. Koch's experimental proof that microorganisms cause disease in the early 1800's, provided the impetus to study methods to grow and characterize harmful, as well as beneficial microorganisms. Koch's early experiments to determine the etiology of infectious diseases, led to methods for cultivation of microorganisms on the surface of solid media (e.g., potato slices, see Koch, “Methods for the Study of Pathogenic Organisms,” in T. D. Brock,
Milestones in Microbiology,
American Society for Microbiology, 1961, pp. 101-108; originally published as: “Zur Untersuchug von pathogenen Organismen,” Mittheilungen aus dem Kaiserlichen Gesundheitsamte 1:1-48 [1881]). These studies eventually led to the development of agar as a culture medium component useful for producing solid media for growing isolated colonies of bacteria. To this day, isolated colonies are required (i.e., “pure cultures”) to biochemically identify organisms.
The field of diagnostic and clinical microbiology has continued to evolve, and yet, there remains a general need for systems that provide rapid and reliable biochemical identifications of microorganisms. In particular, it has been very difficult to develop an identification system which is capable of identifying various diverse types of organisms, ranging from the common isolate
Escherichia coli
to the less commonly encountered actinomycetes and fungi.
Methods and identification systems to characterize microorganisms widely used in industry for production of food and drink (e.g., beer, wine, cheese, yogurt, etc.), the production of antibiotics (e.g., penicillin, streptomycin, etc.), bioremediation of oil spills, biological control of insect pests (e.g.,
Bacillus thuringiensis
), and the production of recombinant proteins, are still needed. In addition, very few identification methods and systems have been developed for environmental use and there remains a need for simple and generally useful identification methods of many organisms. In particular, methods for identification and growth of the actinomycetes are lacking.
I. The Actinomycetes
The actinomycetes (members of the order Actinomycetales) include a large variety of organisms that are grouped together on the basis of similarities in cell wall chemistry, microscopic morphology, and staining characteristics. Nonetheless, this is a very diverse group of organisms. For example, genera within this group range from the strict anaerobes to the strict aerobes. Some of these organisms are important medical pathogens, while many are saprophytic organisms which benefit the environment by degrading dead biological or organic matter. While many of these organisms grow optimally at temperatures common in the environment (e.g., 25-27° C.), some organisms are quite capable of growing at the body temperature of most mammals (e.g., approximately 35-37° C.). However, two genera of medically important actinomycetes (Thermomonospora and Micropolyspora) are true thermophiles, capable of growing at temperatures ranging to 50° C.
Colonies may be bacterium-like (i.e., ranging from butyrous to waxy and glabrous), or fungus-like (i.e., heaped, leathery, membranous colonies that are covered with aerial hyphae). Many actinomycetes exhibit filamentous growth with mycelial colonies, and some actinomycetes cause chronic subcutaneous granulomatous abscesses much like those caused by fungi. Because of these similarities, the actinomycetes were long-regarded as fungi, rather than bacteria (see e.g., G. S. Kobayashi, “Actinomycetes: The fungus-like bacteria,” in B. D. Davis et al.,
Microbiology,
4th ed., J. B. Lippincott Co., New York, 1990), pp. 665-671).
Despite their similarities with the fungi, the actinomycetes have typical prokaryotic characteristics in terms of nucleoid and cell wall structure, antimicrobial sensitivity, the absence of sterols, motility by means of simple flagella, and long filaments of the diameter of bacteria (approximately 1 &mgr;m, compared to the larger fungal hyphae). Microscopically, the morphology of the aerobic actinomycetes varies widely between genera and species, although they are generally observed as gram-positive rods or branching filaments. Some genera never progress beyond a typical bacterium-like coccoid or bacillary form (e.g., Rhodococcus sp.), while others form filaments with extensive branching (e.g., Nocardia, Streptomyces, Actinomadura, and Nocardiopsis). Most are non-motile in their vegetative phase of growth. However, some genera tend to form branched filaments which eventually fragment into motile, flagellated cells (e.g., Oerskovia sp.) (see e.g., G. Land et al.,. “Aerobic pathogenic Actinomycetales,” in A. Balows et al.,
Manual of Clinical Microbiology,
1991, pp. 340-359).
Most of the actinomycetes form spores, with the type of spore formation serving as a phylogenetic and taxonomic tool for separating the organisms into groups. The actinomycetes are highly diverse, with at least ten subgroups. They are also closely related to such organisms as the coryneform group (e.g., Corynebacterium sp.), the propionic acid bacteria (e.g., Propionibacterium sp.), and various obligate anaerobes (e.g., Bifidobacterium, Acetobacterium, Butyrvibrio, and Thermoanaerobacter). The following table lists the organisms included in the suprageneric groups of actinomycetes as set forth in the most recent edition of
Bergey's Manual,
vol. 4, (Stanley T. Williams, editor of vol. 4; John G. Holt, editor in chief,
Bergey's Manual® of Systematic Bacteriology
, Williams & Wilkins, pp. 2334-2338 [1989]).
TABLE 1
Actinomycetes Groups
Representative
Number
Group
Groups/Genera
I
Actinobacteria
Group A: Agromyces,
Aureobacterium
Group B: Arthrobacter, Rothia
Group C: Cellulomonas, Oerskovia
Group D: Actinomyces,
Arcanobacterium
Group E: Arachnia, Pimelobacter
Group F: Brevibacterium
Group G: Dermatophilus
II
Actinoplanetes
Actinoplanes, Ampullariella,
Micromonospora
III
Maduromycetes
Actinomadura pusilla
group,
Microbispora,
Streptosporangium
IV
Micropolysporas
Actinopolyspora, Faenia,
Saccharomonospora
V
Multilocular
Frankia, Geodermatophilus
Sporangia
VI
Nocardioforms
Nocardia, Rhodococcus,
Caseobacter
VII
Nocardioides
Nocardiodes
VIII
Streptomycetes
Streptomyces, Streptover-
ticillium, Kineosporia
IX
Thermomonosporas
Thermomonospora, Nocardiopsis,
Actinomadura madurae
group
X
Other Genera
Glycomyces, Kitasatosporia
Spirillospora, Thermoactinomyces
Although these organisms may often be identified to the genus level based on their morphology at the time of primary isolation, organisms that have been repeatedly transferred in the laboratory often do not retain their typical morphologic characteristics and must be identified biochemically, or by analysis of their membrane fatty acid composition. Serological methods for identification and differentiation are rarely used, due to the extensive degree of cross-reactivity among the actinomycet

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