Broad spectrum fastidious organism culture medium

Details Broad spectrum fastidious organism culture medium Broad spectrum fastidious organism culture medium

2002-05-14

2003-04-29

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S032000, C435S014000

Reexamination Certificate

active

06555333

ABSTRACT:

BACKGROUND OF THE INVENTION
The use of antimicrobial therapy to treat bacterial infections and diseases in humans and animals is well known throughout the world. Successful therapy requires selection of an antimicrobial agent that is specific to the bacteria causing the infection or disease. This antimicrobial agent selection is typically made by the health practitioner with the assistance and input of clinical microbiological laboratories which can measure the in vitro susceptibility of bacteria to antimicrobial agents by a variety of methods.
In many such laboratories a standard method of assessing the susceptibility of common pathogens to antimicrobial agents is by agar disk diffusion, commonly known as Disk Susceptibility testing, whereby a disk impregnated with an antimicrobial agent is pressed onto agar that has been infused with the suspected pathogen that has been isolated from a specimen taken from the patient, the agar is incubated and microbial growth or inhibition around the disk is observed and recorded. The National Committee for Clinical Laboratory Standards (NCCLS) has formulated specific uniform methods for such Disk Susceptibility testing, criteria for quality control testing and tables for quantitatively measuring the degree of pathogenic bacteria inhibition. See “Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Seventh Edition,” NCCLS Bulletin M2-A7 (January 2000).
Many common pathogens are “fastidious” microorganisms in the sense of requiring a special culture medium and a controlled environment to grow. The NCCLS Bulletin recommends the use of a Gonococcal (GC) agar medium supplemented by a 1% defined growth supplement for Disk Susceptibility testing of the fastidious microorganism
N. gonnorrhoeae.
See NCCLS Bulletin M2-A7 at page 10 and Jones et al., 27
J. Clin. Microbiology
2758 (1989). However such a supplemented GC agar medium was not recommended for use with any other fastidious microorganisms. The NCCLS Bulletin recommends that a variety of culture media having different formulations be used to test fastidious microorganisms such as Haemophilus species,
N. gonorrhoeae, Streptococcus pneumoniae, Streptococcus viridans
and &bgr;-hemolytic Streptocci. Ibid, page 9. This requires the preparation of a different culture medium for each suspected pathogen, a cumbersome and time-consuming task when, as is usually the case, the identify of the pathogen is unknown.
There is therefore a need in the art for a single culture medium suitable for antimicrobial Disk Susceptibility testing for multiple fastidious microorganisms and even simultaneous testing for such microorganisms.
BRIEF SUMMARY OF THE INVENTION
The foregoing need is met by the present invention, which provides a culture medium suitable for simultaneous antimicrobial Disk Susceptibility testing of multiple fastidious microorganisms, including Haemophilus species,
Streptococcus pneumoniae, Streptococcus viridans
species, &bgr;-hemolytic Streptocci, Neisseria species and Campylobacter species.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention lies in the discovery that the further supplementation of a particular known supplemented GC agar medium provides a culture medium useful in Disk Susceptibility testing for multiple fastidious pathogenic microorganisms.
The known supplemented GC agar medium (hereafter referred to as “base GC medium”) comprises a mixture of deionized water, agar, peptones, beef and yeast extracts, starch, glucose and phosphate buffer components. More specifically the non-aqueous portion of this base GC medium comprises the components noted below present in the amounts noted:
component
wt % (±5%)
agar
30
casein peptone
15
meat peptone
8
proteose peptone
5
peptonized milk
4
beef extract
3
yeast extract
3
starch
3
glucose
1
NaCl
14
K
2
PO
4
10
KH
2
PO
4
2
Na
2
CO
3
1
The foregoing non-aqueous portion is preferably mixed with deionized water in a concentration of about 40 g/L of water.
The deionized water may have a pH of 5 to 7, preferably 7, since the final pH of the culture medium of the present invention is preferably 7.3±0.1 at ambient temperature; its bacteriological purity should be less than 100 CFU/mL with a TOC content of less than about 712 ppb and a resistivity of ≧2 megohms.cm at 25° C. The agar used in the base GC medium is preferably of the Iber type derived from Gelidium seaweeds that is of pharmaceutical grade, commercially available from DMV International Nutritionals of Fraser, N.Y. (hereafter “DMV”).
The remaining components of the base GC medium and their commercial availability is as follows: casein peptone, a pancreatic digest of the complex of milk proteins known by that name, available as Product No. CE90-M from DMV; meat peptone, an enzymatic digest of animal tissue that is preferably a 50/50 mixture (w/w) of Product No. 102, available from Global and Product No. AE80M, available from DMV; proteose peptone, available as Product No. PP90M from DMV; peptonized milk, available as Product No. 5X59048 from Quest International Co. of Norwich, New York; beef extract, available in powder form as Product No. 150 from Global BioIngredients of Tampa, Fla. (hereafter “Global”); yeast extract, available as Product No. 151 from Global; starch (preferably reagent grade potato starch) available as Product No. S-123 from Pfanstiehl Laboratories, Inc. of Chicago, Ill.; and glucose, available as Product No. DE140 from Spectrum Laboratory Products, Inc. of Gardena, Calif. (hereafter “Spectrum”). The remaining chemical components of NaCl, K
2
HPO
4
, KH
2
PO
4
and Na
2
CO
3
are readily commercially available from many sources, and are preferably of ACS reagent grade.
Further supplementation of the foregoing base GC medium with a multi-component amino acid supplement and with laked horse blood allows its use for antimicrobial Disk Susceptibility testing for at least six classes of fastidious microorganisms, including Neisseria species, Haemophilus species,
Streptococcus pneumoniae, Streptococcus viridans
species, &bgr;-hemolytic Streptocci and Campylobacter species.
The amino acid component comprises deionized water and L-cysteine HCl, L-cystine, L-glutamine, guanine HCl, thiamine HCl, p-aminobenzoic acid (PABA), vitamin B12, cocarboxylase, nicotinamide adenine dinucleotide (NAD); adenine, glucose, HCl and Fe(NO
3
)
3
.9H
2
O (ferric nitrate nonahydrate).
In a preferred formulation the amino acid component comprises an aqueous solution of the following components in the following approximate concentrations
con-
component
centration
L-cysteine HCl
26
g/L
L-cystine
1
g/L
L-glutamine
10
g/L
guanine HCl
.03
g/L
thiamine HCl
.003
g/L
PABA
.01
g/L
vitamin B12
.01
g/L
cocarboxylase
0.1
g/L
NAD
.25
g/L
adenine
1
g/L
glucose
100
g/L
Fe(NO
3
)
3
· 9H
2
O
.02
g/L
HCl(12N)
5
mL/L
The non-aqueous components of the above amino acid component are commercially available as follows.
Component
Product No.
Source
L-cysteine HCl
CY115
Spectrum
L-cystine
CY120
Spectrum
L-glutamine
GL136
Spectrum
guanine HCl
G6377
Sigma
(St. Louis, MO)
thiamine HCl
T4625
Sigma Chemical Co.
PABA
AM150
Spectrum
vitamin B12
CY105
Spectrum
cocarboxylase
C8754
Sigma
NAD
N1102
Spectrum
adenine
0183
Amresco, Inc.
(Solon, OH)
glucose
DE140
Spectrum
Fe(NO
3
)
3
· 9H
2
O
F1030
Spectrum
The amino acid component is preferably prepared by adding the L-cysteine HCl and L-cystine components to the deionized water in powdered form and stirring while adding the 12N HCl until the two powdered components are dissolved. The guanine HCl is added to the resulting solution and stirred for about 30 minutes until the same is dissolved. The remaining amino acid component ingredients are then added and the solution stirred until all components are in solution. The solution is then filtered with a 0.2 micron filter into a container that has been autoclaved at 127° C. for at least 30 minutes.
The laked horse blood component preferably comprises lysed red blood cells extracted from defibrinated horse blood. A preferred method of lysing and extraction is to conduct

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