CA1118328A

CA1118328A - Device for detecting deoxyribonuclease production

Info

Publication number: CA1118328A
Application number: CA000325042A
Authority: CA
Inventors: Christopher Hill; Mau H. Kuo; Devendra V. Mehta
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1978-05-01
Filing date: 1979-04-06
Publication date: 1982-02-16
Also published as: IT7948891A0; IT1116469B; SE7903639L; JPS54145596A; GB2020017A; GB2020017B; DE2914544A1; FR2425073A1

Abstract

ABSTRACT
DEVICE FOR DETECTING DEOXYRIBONUCLEASE PRODUCTION

A test device, process for making the device and method of determining the production of deoxyribonuclease in a microbiological sample are disclosed. More parti-cularly, a test device is provided for determination of deoxyribonuclease production in a microbiological sample, which device comprises a carrier matrix and, incorporated therewith, deoxyribonucleic acid, methyl green and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose.

Description

Docket No. 11594 DEVICE FOR DETECTING DEOXYRIBONUCLEASE PRODUCTION

The present invention relates generally to the field of microbiological test devices and, more particularly, to - 5 a test device, process for making the device and method of determining the production of deoxyribonuc]ease in a microbiological sample.

BACKGROUND OF THE INVENTION

; The ability of an organism to break down deoxyribo-nucleic acid (DNA) by means of a deoxyribonuclease (DNAase) has been used clinically to differentiate between groups of microorganisms [Rothberg, N.W. et aZ. J. Baot., 90:294-295 fl965~; BZa~evic, D.J. et aZ. Am. J. CZin. PathoZ. 51: 277-279 fl9ff9); Mart~n, W.J. et aZ. Canad. J. MiorobioZ., 13:
15 616-618 (1967~], to aid in determining the potential pathogenicity of staphylococci lWeekman, B.G. et aZ. J.
Baot., 73:7~7-753, (1957)], and to determine the immuno-logical response to group A streptococcal infections [Wannamaker, L.~., In Vhr, J.W., Ed. The Strepto¢ocous, Rheumati¢ Fever, and GZomeruZonephritis, WiZZiam~ B ~iZkins, BaZt~more (1965)].
DNA is a polynucleotide that is composed of a chain of deoxyribonucleotide units. Deoxyribonucleotides are , phosphoric acid esters of nucleosides (a pyrimidine or purine base attached to deoxyribose). The phosphoric ~L

:
,. . . .
, .
., ; .

1~L18328 acid is attached to the deoxyribose ring at either the 3 or 5' position. Examples of deoxyribonucleotides are adenosine-5'-monophosphate (5'-AMP) and guanosine-S'-mono-phosphate (5'-GMP~.
DNAase is an extracellular enzyme that hydrolyzes DNA
to yield oligonucleotides (chains of several deoxyribo-nucleotides). The DNAase of Serratia mar~e6cens yields di-, tri-, and tetranucleotides [NestZe, M. et aZ. J.
BioZ. Chem. 244:5219-5225 (1969) 3. The DNAase of Group A
streptococci produces predominantly products having a chain length of 3 to 10 deoxyribonucleotide units, but also produces mononucleotides [Wannamaker, supra].
DNA has physical and chemical properties different from those of oligonucleotides or mononucleotides, and advantage has been taken of these differences to detect the hydrolysis of DNA by DNAase. Not all of the many methods proposed to determine hydrolysis of DNA are applicable in the clinical laboratory.
An improved medium for detecting DNAase production has been developed [Smith, P.B. et aZ. AppZ. M~crobioZ.
18:991-993 fl969)]. This method utilized a DNAase test medium to which is added methyl green. Methyl green com-bines with DNA to form a green complex at pH 7.5. If the DNA is hydrolyzed, the methyl green is released and be-comes a colorless compound [Kurnick, N.B. et aZ. Arch.
Biochem. 29;41-53 fl950)]. Therefore, the uninoculated medium with DNA is green, if an organism produces DNAase a colorless zone will be seen around the colonies. Gram-positive organisms grow well on this medium.
; 30 Most of the bacterial DNAases studied have been found to require the presence of divalent cations for their activity [Wannamaker, supra]. Media containing peptones will usually contain th~se cations. The pH optima have also been investigated and have been found to range from 5.5 to 8. 5 [Wannamaker, supra; NestZe~ M. et aZ. J. BioZ.
Chem. 244:5213-5218 rl969)]. For clinical purposes, media with a pH of 7.2 have been found to be satisfactory.

.

In another line of investigation, methods have recently been devised in which the standard nutrient medium plate or tube is replaced by nutrient impregnated pads Such devices may comprise a bibulous material such as paper into which the nutrient medium is incorporated in a dried state. These devices are usually employed as test devices by reconstituting the dry media with a sterile fluid, streaking or otherwise contacting the surface of the device with the fluid specimen to be tested, incubating the same under standardized conditions and observing the growth of microorganism locations on the surface. Exemplary of such devices are those disclosed in U.S. Patents Nos.
3,699,003 and 3,881,993.
Freake, U.S. Patent No. 3,881,993, discloses a test device for analyzing a sample for microorganisms, said device being adapted to be inoculated with the sample and incubated in a sealable container, the device comprising an absorbent matrix impregnated with a nutrient medium, a reagent, and a water soluble culture-fixing agent capable of forming a ~iscous suspension in an aqueous solution to localize the microorganisms on said matrix.
Kronish, U.S. Patent No. 3,699,003, discloses a pre-paration for the biochemical identification of ~-hemolytic streptococci into serologic groups, e.g., groups A, B, C, D, etc. This preparation comprises a carrier, e.g., a bibulous strip containing a glycerol reagent band, a ; salicin reagent band and an esculin reagent band. In the glycerol and salicin reagent bands there are also pH
indicator systems. The esculin band contains a compound which reacts with esculin breakdown products to produce a color change. These bands are separated from each other by hydrophobic barriers. In use, a quantity of the micro-organism whose serological identification is to be deter-mined is transferred onto each of the three reagent bands.
Notwithstanding these contributions by prior workers in the field, there has been no quick and con~enient means for determinations of deoxyribonuclease production by microbiological samples.
,.......................................... .

, Z.

.
.

~83~8 OBJECTS OF THE INVENTION

It is therefore an object of the present invention to provide an improved test for the detection of deoxyribo-nuclease in biological samples.
It is another object of the invention to provide an improved deoxyribonuclease test in device format.
Yet another object of the invention is to provide a test device for detection of deoxyribonuclease which can be used directly for collecting the biological specimen.
1~ It is therefore a further object o the present invention to provide a test device, having the above-mentioned advantages, comprising an absorbent matrix incorporated with deoxyribonucleic acid, methyl green, and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose.
Other objects and a fuller understanding of the invention will be had by referring to the following descrip-tion and claims drawn to preferred embodiments thereof, taken in conjunction with the accompanying drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a plan view of a test device according to the invention having deoxyribonuclease nonproducing colonies thereon; and Fig. 2 is a plan view of a test device according to ; the invention having deoxyribonuclease producing colonies thereon with resultant plaque or clear zone formation.

SUMMARY OF THE INVENTION
.
In accordance with the present invention there is provided test means for determining the production of deoxyribonuclease in a microbiological sample. More particularly, a test device is provided for the detection of deoxyribonuclease production by a microbiological 33~8 sample which comprises a carrier matrix and, incorporated therewith, a composition comprising deoxyribonucleic acid, methyl green, and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose. Incorporation of the deoxyribonuclease detection system into the carrier matrix is accomplished by impregnation of the carrier matrix with an impregnating solution containing the detection system. A range for the polymer concentration in the impregnating solution is advantageously from about ~.5 grams/ deciliter (g/dl) to about 3.5 g/dl, with

2.0 g/dl to 3.0 g/dl, and especially 2.6 g/dl, being preferred.
Presumptive identification of ~-hemolytic strepto-coccal strains, assumed to be pathogenic and from Lancefield group A, is possible by addition of an appropriate selec-tion of microbial inhibitors in satisfactory concentrations to devices in accordance with the invention. The sub-stantial clinical significance of early and convenient identification of these widespread and invasive pathogens can be readily appreciated.
As an additional aspect of the invention there is provided a process for the determination of deoxyribo-nuclease production by a microbiological sample which comprises contacting a sample with the aforementioned devices, incubating the devices so contacted with the sample, and observing any plaques formed thereon.
The devices described in accordance with the inven-tion are prepared by a process of incorporating an absor-bent matrix with a composition comprising deoxyribonucleic acid, methyl green, and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose.
It is now possible by using the test device of the present invention to perform in a rapid and convenient manner a determination of clinical specimens, such as body fluids, for the presence of organisms which produce deoxyribonuclease, Such organisms include various aspects of streptococcus, staphylcoccus and organisms in the family Enterobacteriaceae.
.

~1~83~Z~3 DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to Fig. 1, the test device of the present invention, generally designated as 1, essentially com-prises an absorbent matrix 3, and incorporated therewith, a composition comprising deoxyribonucleic acid, methyl green and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose. MicrobiaI colonies 2 are interspersed throughout and are visible from the surface of matrix 3. Depicted in this illustration is a reacted device indicating the organism cultured thereon to be a nonproducer of deoxyribonuclease. This is illustrated by noncleared zone 4 which is the unaffected area of the impregnated matrix adjacent to individual colonies 2 growing thereon. Such an appearance of a reacted device would clinically be considered a negative response.
Fig. 2, illustrating a DNAase positive response, shows microbial colonies 2 around which there is a clear zone 5, indicating detection of and reaction with deoxy-ribonuclease by the test composition incorporated into matrix 3. The mechanism of the reaction has in the past generally been considered to be one wherein methyl green combines with polymerized DNA to form a stable, colored complex. If the DNA is hydrolyzed, such as by deoxyribo-nuclease, the methyl green is freed and fades to a color-less compound, thus resulting in the appearance of plaquesidentified on the matrix of the in~ention as clear zone 5.
For the purpose of the present disclosure the term "microorganism" refers to the class of microorganisms including yeast or other fungi, mycoplasma, pleuropneumonia-like organisms, rickettsiae, and chlamydia, but particu-larly to bacteria. The term "colony" refers to the "microorganism" subsequent to culturing. The expression "colony location" or "microbial colony" indicates the site of a colony on matrix 3.

~1~832~

The expression "carrier matrix" or the term "matrix"
refers to bibulous matrices which are insoluble and main-tain their structural integrity when exposed to physio-logic:al or other liquids. Suitable bibulous matrices which can be used include paper, cellulose, wood, synthetic resin, fleeces, non-woven and woven fabrics and the like.
For convenience the matrix can be associated with an insoluble support member, such as one made of polystyrene.
The invention further provides a method for using the test device for determination of deoxyribonuclease pro-duction in microbial test samples The sample is tested by contacting the device with the sample, incubating the device so contacted under generally physlological tempera-ture parameters and observing any clear zone formation on the device after a suitable incubation period. Incubation for the present device is characteristically for a period of from about 12 to about 48 hours, preferably at a temperature generally in the range of 37C.
DNAase test agar used in the following examples were obtained from Difco Laboratories, Detroit, Michigan. Calf thymus DNA used in the following examples was obtained from Research Products Division, Miles Laboratories, Inc., Elkhart, Indiana. Methyl green used in the following examples was obtained from Fisher Scientific Co., Pittsburgh, Pennsylvania and was their M-295 certified biological stain. Standard reagent grade polyvinyl alcohol, carboxy-methylcellulose, and polyvinylpyrrolidone were also obtained from Fisher Scientific Co.
The following examples are merely illustrative and are not to be construed as a limitation of the invention.
One skilled in the art will be able to make such variations, substitutions and changes in the ingredients and para-meters as may seem desirable.
.
EXAMPLE I

Test devices incorporating the deoxyribonucleic acid/
methyl green test for detection of deoxyribonuclease pro-^~ duction were prepared with various additives and compared

3~8 for production of a distinct clear zone surrounding the colony, indicating deoxyribonuclease production.
Solutions according to various formulations were pre-pared for impregnation of paper matrices A base solution was prepared in 300 milliliters (ml) of distilled H20 by addition thereto of 1.50 grams (g) of NaCl, 0 18 g of MgS04, 0 6 g deoxyribonucleic acid (DNA), 0 30 g methyl green and 6.0 g tryptose. The base solution was divided into five aliquots of 50 ml each.
Each of four aliquots of base solution, as prepared above, then received a different additive as follows:
(1) Carboxymethylcellulose (CMC) 1 g ~2) Polyvinyl pyrrolidone ~PVP) 1 g (3) Polyvinyl alcohol (PVA) 1 3 g ~4) Agar 0.5 g (5) Sodium alginate 0 25 g A control was run which contained 50 ml of the base solution, as such, The solutions containing additives were mixed over-night at room temperature. The following day the solu-tions and control were heated to a boil and then auto-claved for 15 minutes at slow exhaust at 121 Centigrade ~C) Sheets of Schleicher ~ Schull (S ~ S) 470 filter paper ~Schleicher ~ Schull Co.~ Keene, NH) were separately impregnated to saturation with each of the solutions and the control, respectively, allowed to dry at room tempera-ture and cut to 2,5 centimeters ~cm) x 2 5 cm to provide test devices. The devices were then placed into petri dishes.
The devices were inoculated with a known culture of ~-hemolytic streptococci species at concentrations of 103/ml, 105/ml and 106/ml organisms. The devices were then incubated for 24 hours at 37C.
The devices prepared with formulations having poly-vinyl alcohol and those having carboxymethylcellulose had distinct "clear" or decolorized areas surrounding colonies of deoxyribonuclease-producing microorganisms at all microorganism concentrations tested. The area surrounding ill83Z8 g the clear zones remained as unchanged shade of green.
Those devices wherein the polymer polyvinylpyrrolidone was used did not, however, provide satisfactory distinct clear zones.
Agar impregnated into the paper only slightly and with difficulty. It tended, rather than impregnating, to form a film layer on the paper. In those devices in which it was possible to impregnate the agar, a diffuse uniform fading of green towards a yellowish color was observed with no distinction around colonies. Although impregna-tion of the paper with formulations having sodium al~inate was not as difficuit as with the agar, the results observed, uniform blanching, were very similar to the agar and, thus, equally unacceptable.
Overall performance indicated PVA to be the best additive for providing distinct clear zones in the test device, with carboxymethylcellulose additionally pro~iding visibly distinguishable clear zones around the colonies.
;

EXAMPLE II

Testing was undertaken to determine the optimum con-centration of PVA for reproducible, distinct zones of clearing, indicating the presence of deoxyribonuclease.
Devices were prepared as in Example I using seven different impregnating solutions respectively containing PVA in the following concentrations: 0.5 grams/deciliter (g/dl), 1.0 g/dl, 1.5 g/dl, 2.0 g/dl, 2.6 g/dl, 3.0 g/dl and 3.5 g/dl.
These device~ were inoculated with the streptococcus organisms from the same culture and in the same concen-trations as used in Example I and incubated as describedtherein.
A distinguishable clear zone around the colony forma-tions was observed at all PVA concentrations tested. The sharpness of demarkation between areas of clearing and the ; 35 green unaffected areas was optimized in the device prepared with the impregnating solution in which PVA was present at the 2.6 g/dl concentration level.
i . .
' ~ ~
:

~118328 Thus, a range of acceptable polymer concentration inthe impre~nating solution of from about 0,5 g/dl to about 3,5 g/dl was shown to be functional in the invention, , EXA~IPLE III

Te t devices incorporating the deoxyribonucleonic aci~s/methyl green test of the present invention are provided ~ith selective chemical inhibitors and anti-biotics so as to provide a de~ice capable of presumptively identifying ~-hemolytic streptococci, Six separate impregnation solutions were prepared in S0 ml of distilled H2O by addition thereto of 0,25 g of NaCl, 0,03 g of ~gSO4, 0,1 g of DNA, 0,05 g of methyl green, l,O g of tryptose, and were formulated to be selective for ~-hemolytic streptococci by addition thereto of one of the following selective chemical inhibitors:
crystal.violet 50 milligrams (mg) (staphylococcus inhibitor) amphotericin B 0,25 mg (mycological inhibitor) sodium colistimethate 2 mg (gram negative inhibitor) naladixic acid 1~5 mg (gram negative inhi~itor) gentamycin 25 micrograms (~g) .
' 25 (staphylococcus inhibitor) `. lithium chloride 0,18 mg (a-hemolytic str~ptococcus) Each impregnation solution was mixed overnight at room temperature. The following day each solution was heated to a boil and then autoclaved for 15 minutes at . slow exhaust at 121C. Separate sheets o~ S ~ S 470 filter paper were respectively impregnated to saturation with the impregnation solutions, allowed to dry at room temperature and cut to 2.5 cm x 2,5 cm to provide devices according to the invention, The devi.ces were then placed into petri dishes, .

': .

, ~118328 One set of devices prepared as above was inoculated with a known culture of ~-hemolytic streptococcus species at concentrations of 103/ml, 105/ml and 106/ml organisms.
Another set of devices prepared as above was inoculated with similar concentrations of DNAase producing staphylo-coccus strains, and a third set of such devices was inoculated with similar concentrations of DNAase producing gram negative organisms. The devices were then incubated for 24 hours at 37C.
The devices were observed after incubation, and clear zone formation was found to be present only in the devices inoculated with the ~-hemolytic streptococcal strains.
Growth was not observed on the other devices and clear zones, indicative of DNAase production, therefore, did not appear.
As can be seen from these results, incorporation of inhibitors into devices in accordance with the invention does provide in addition to a convenient device for deter-mination of DNAase production in strip format, a ~evice which offers presumptive identification of ~-hemolytic streptococcus organisms, such as from clinical samples.
Becuase of the wide spread occurrence of these pathogenic organisms, such a device clearly has distinct advantages for clinical laboratory use.
Although the invention has been described with a certain degree of particularity, it is understood that the present disclosure has been made only by way of example and that numerous changes may be resorted to without departing from the spirit and scope of the invention.

.

~:,

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A test device for determination of deoxyribo-nuclease production in a microbiological sample, which device comprises a carrier matrix and, incorporated there-with, deoxyribollucleic acid, methyl green and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose.

2. The test device of Claim 1 wherein the deoxyribo-nucleic acid, methyl green and polymer incorporated with the matrix is the dried residue of a previous impregnation of the matrix with a solution thereof.

3. The test device of Claim 2 wherein the polymer is present in the impregnating solution at a concentration of from about 0.5 g/dl to about 3.5 g/dl

4. The test device of Claim 3 wherein the polymer is present in the impregnating solution at a concentration of about 2.6 g/dl.

5. A process for the determination of deoxyribo-nuclease production in a microbiological sample which comprises contacting the sample with the device of Claim 1, incubating the device so contacted, and observing any clear zones formed thereon.

6. The device of Claim 1 which further comprises inhibitors effective to allow selective growth of .beta.-hemolytic streptococci.

7. The device of Claim 6 wherein said inhibitors include crystal violet, amphotericin B, sodium colisti-methate, naladixic acid, gentamycin or lithium chloride.

8. A process for making the device of Claim 1 which comprises incorporating a carrier matrix with a compo-sition comprising deoxyribonucleic acid, methyl green, and a water-soluble polymer selected from polyvinyl alcohol and carboxymethylcellulose.