Note: Descriptions are shown in the official language in which they were submitted.
AHP-6781
Backaround of the Invent on
a) Fieid of the Invention
This invention relates to a method, and an apparatus and
~` test elements for use therewith, for the rapid determination of the
susceptibility of bacteria to antibacterial agents. The method
correlates to the art accepted standards of the Kirby-Bauer method
for the sensitivity testing of pathogenTc bacteria to antibacterial
agents.
b) DescrlDtion of the Prior Art
In the past, varlous methods have been used to determine
anttblotlc sensltlvlty; for example, agar diffusion and serial
dllutlon. As a result, the correlat10n of sensitivity testing results
from vartous hospltals was dlfficult. In 1966, A.W. Bauer et al.,
Amerlcan Journal of Cltnlcal Pathology, 45, 493(1966), reported a
standardlzed dlsc procedure in an attempt to allevlate this sltuation.
In 1972, the FDA publlshed 1n the Federal Register, 37, No.l91,
20527~1972), a notice that the latter standard7zed disc procedure,
whtch ts hereln refered to as the Klrby-Bauer method, Is to be used
as the standard method tn ~11 cllnl CD I laboratorles for the sensltivlty
testlng of pathogenlc bacterla to antlb~cterlal agents. However, the
Klrby-i~uer method ls not wlthout certatn dlsadvantages. Some of the
dlsadvantages are: fallure to use Mueller-Hlnton agar, fallure to
measure the pH of the medlum, Inadequate standardlzatlon of broth
culture denslty, fallure to press surplus fluld from swab before
Inoculatllng,plates, excess delay between culture standardization and
plate InoculatTon, excess delay in applying the dlsc after Inoculation
of plates, wrong Incubatlon temperature and tlme and fallure to measure
.; .
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AHP~
zones carefully. If the Kirby-Bauer method is conducted as outlined
- in the Federal Register with the errors listed above being eliminated,
then any new method and apparatus can be justified only after
experimental data has demonstrated that the aIternate method is at
least as accurate and precise as the recommended Kirby-Bauer method.
Although thTs accepted standard has been established for
the determTnation of bacterial sensitivity to antibacterial agents, the
prlor art does not solve a numb-r of problems. For example, the existing
; apparatus and methods for performing the KirbyC~auer test are noteasily standardlzed to glve conslstent results. In addition, a skllled
technlclan Is necessary for the operatlon of the apparatus and methods.
Another difflculty encountered in the prior art is that the determination
of the bacterial sensitTvTty to the antibacterial agent requires various
measurements and suTtable standard bacterTal strains and the InterpretatTon
Ts only qualltatTve. Thus, a method to gTve an easTly measurable and
dTstTnct end potnt for determTnTng the bacterTal sensTtivity to the
antlbacterTal agent Is clearly needed.
An attempt to solve thls latter problem of correlDtlng th0
bacterlal sensltlvlty to the antlbacterlal agent Is reported by Y. Kanazawa
and T. Kuramata, The Journal of Antlblotlcs, Ser. A, XIX ~5), 229(1966).
In thls report they used oxTdatlon - reductlon dyes or redox indlcators
to glve an Indlcatlon of bacterTal sensltTvTty to the antTbacterial agent.
However, a number of problems remalned unsolved; for example, the malntenance
; of the antTbacterTal agent Tn the dTsc and suTtable concentrations of the
redox Indlcator and antTbacterTal agent to give a correlation of results
wTth an accepted standard.
An obJect of the present Inventlon Ts to provTde an apparatus,
test element and method for the rapid determTnatTon of bacterial
.
~r1r~
14~1
susceptibility to antibacterial agents, the method giving results
equivalent to those of the accepted Kirby-~auer method.
Another object of the present invention is to provide an -
apparatus that gives easily measurable and distinct end points for
determining the susceptibility of bacterial pathogens to all useful
antibacterial agents.
Still another obJect of the present invention is to provide
a new and improved method that is rapid, simple and requires
inexpenslve equlpment and supplies for determtning the suscepttbtItty
of bacterlal pathogens to all useful anttbactertal agents.
Summ ~ entlon
According to one aspect of the present invention, an apparatus
is provlded for the indlcation of bacterial susceptibtlity to anttbactertal
agents. The apparatus Includes a tray dlvlded tnto a plurallty of
compartments, each compartment contalnlng a pad formed from inert material.
A redox Indtcator that changes c'olor In the presence of growlng bacteria
and a particular antibacterial agent is tmpregnated In each pad. The
tray Is provlded wlth a removable tray cover In order to protect and
contaln the pads In the tray. The tray or tray cover can Include
Indlcla to enable a determlnatlon of the character of the antlbactertal
agent In the partlcular compartment.
Another aspect of thls Inventlon Involves a new method for
obtalnlng results equlvalent to those obtalned by the Kirby-i3auer method.
General DescrlPtlon of the Inventlon
The term "antlbactertal agent" as used heretn tncludes anti~
bacterlal, antlblotlcs and synthettc antibactertal agents.
The efflcacy of the anttbacterial agents ts evident by
observation of the color change of the pad. A color change tndicates that
the organlsm grows In the presence of the anttbacterial agent in the pad
; ~ AHP-6781
14i~
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and is not susceptible to it, whereas if the pad does not change color
the organism does not grow and is susceptible to the antibacterial agent
in the pad. The pads are standardized so that the color change and the
lack of color change indtcate reslstance and susceptibility of the
bacterium,-rospectlvely, and correlate to the interpretatlon of the accepted
Klrby-Elauer method tt.e., to the standards as publlshed in the Federa:
RegTster, clted above).
The nature of the apparatus lends Itself to almost complete
standardlzatlon so that ITttle Is left for the technlclan to do but add
a standard volume of the standardlzed Inoculum of bacteria under test
and Incubate the Inoculated apparatus. The characteristlcs of the test
; element also provide the apparatus or kit wtth a very practical shelf-life.
The present apparatus is an tntegrated, relatively tnexpenslve
package, unlquely adapted for standardlzatlon and can be discarded
'5 upon completlon of the test. The apparatus thus affords a rapid and
practlcal means for determlnlng the suscept7billty of bacterla to
particular antlbacterlal agents. The redox Indlcator, the antlbacterlal
agents and thelr ~rrangement are standardlzed and are an Integral part of the
of the apparatus. All that the user needs to do Is add a standardlzed
inoculum of the b~cterlum under test dlrectly to each pad contalning
a glven antlbacterlal agent and examlne each pad for color change after
the speclfled perlod of Incubatton.
Sultable Tnert materlals for the pad are selected from
porous or flbrous materlals havlng an absorptlve property. Examples of
such Inert materlals Include alpha cellulose paper (I.e. fllter paper),
cellulose acetate and polyurethane foam. The Inert materlal must be
clean and contaln no substances whlch will Inhiblt or enhance the
actlvlty of the antlbacterTal agent absorbed thereln, nor affect the pH.
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~ AHi~6781
14~)
The color of the inert materi~l can vary; however, the color selected
should not interfere with the detection of the color change of the dye.
The shape, sTze and thickness of the pad are not critlcal and can be
varled so long as the pad contains the required amount of redox
indicator and antTbacterTal agent, and wlll absorb the standardized
tnoculum. The pads can be preformed and impregnated by conventional
means. A convenlent means includes the use of an inert, volitile liquid
carrler for the impregnation. Alternatively, the redox indicator and
antibacterlal agent are absorbed into a sheet of the tnert materTal,
followed by cuttlng out the pads from the sheet. A useful and preferred
Inert materlal pad Is the 0.5 Inch dlameter, 0.034 Inch thlck,
hlgHly absorbent paper dlsc No.740-E, ava11able from Schelcher and
Schnell Inc., Keene, New Hamshlre.
~arlous redox Indlcators can be absorbed Into the pad,
~5 for example resazurln, trlphenyltetrazollum chlorlde, 2,6-dichloro-
phenollndophenol and the llke. Requlrements for selecting a redox
Indlcator dye are that the redox Indlcator must be responslve to changes
In the redox potentlal whlch re9ult from met~bollc processcs of llvlng
bacterla and that the response, once It has taken place, is not
reverslble. A sultsble range of redox potentlal Is an oxldatlon-
reductlon potentlal between +0.2 and +0.05 volts. In addltlon, the
redox Indicator should not Inhlblt or enhance the actlvlty of the antl-
bacterlal agent absorbed theretn, act on the bacterlum under test nor act
as pH Indlcator, for example In the pH range of from 6.0 to 8Ø The
amount of the redox Indlcator absorbed Into the pad Is selected so that
the redox Indlcator changes color from the metabollc processes of
llvlng bacterla wlthln a perlod of 3 to 36 hours, preferably 6 to
24 hours, at about 37~C.
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AHP-6781
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A preferred redox Indicator satTsfylng these requirements
is resazurin ~7-hydroxy-3H-phenoxazin-3-one 10-oxide). Resazurin (blue)
is reduced to resorufin (pink) at an oxidatton - reduction potential
between +0.2 and +0.05 volts and this phase of reductlon is not
reversible. The concentration or amount of the redox indlcator present
fn each pad Is dependent upon whether gr~m posi+ive organisms or gram
negative organtsms are to be tested. For example, when resazurin
Is used as an indicator of bacterial growth, the recazurin at a
concentratlon of I to40 ~g, preferably 2 to 4 ~9, per 0.5 tnch diameter
paper dlsc ~descrlbed above) glves a vlslble color change of the
Indlcator ~blue ' plnk) wlthln a slx hour poriod of Incubation at
37C wlthout a subsequent change after 24 hours Incubatlon at 37C for
gram posltlve bacterta. For gram negatlve organlsms, a concentration
of l.Oto 100 1l9, preferably 10 to 20 ~9, per the 0.5 Inch dlameter
paper dlsc Is requlred to obtaln a vlslble color change of the
Indlcator ~blue plnk) wlthln a slx hour perlod of Incubatlon at 37C
wlthout any subsequent change after 24 hours incubation at 37C.
Vlrtually any of the avallable ~ntlbacterl~l agents can
be Impregnated In the p~ds. Examples of these antlbacterlal agents
are as follows: the penlcllllns, exempllfled by penlclllln G,
penlclllln V, amplclll1n, cloxaclllln, dlcloxac~llln, methlclllln,
c~rbenlclllln and the llke; the cephalosporlns, exemi~llfled by
cephalosporln C, cephalothln, cephalorldlne, cephaglycln, cephalexin,
cefazolln, cefamandolo, cefoxltln and the llke; the sulfa drugs
exempllfled by sulfathlazole, sulfaguanldlne, sulfamethizole,
sulfamethoxazole and the llke; the tetracycllnes, exempllfled by
tetracycllne, chlorotetracyclln-, oxytetracycllne and the llke; the amino
glucosldes, exempllfled by streptomycln, neomycln, gentamlcin and the
llke; mlscellaneous antlbacterlal agents, ex-mpllfled by novoblocin,
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AHP-6781
colimycln, lincomyctn, trlmethoprim, clindomycin, erythromycin,
chloramphenicol, nitrofuranto7n, furazol1done, nalidixlc acid,
polymyxin ~, kanamycin and the like; combtnattons of antTbactertal
agents, exempltfled by a combtnatlon of sulfamethoxazole and trtmethoprim
~i.e., i3actrlm~ and the llke; In thelr pharmaceutlcally acceptable
forms. "In thelr pharmaceutically acceptable forms" refers to the
form In whlch such antlbacterlal agents are admlnlstered therapeutically;
for example, alkall metal salts thereof such as cephslothtn sodium,
cefazolin sodlum, potasslum penlcllltn G, and the Itke, the ammontum
salt of cefamandole, Internal salts such as the zwltter tons of
amplclllln, cephalogiycln and cephalextn, and the betaln of cephaloridine.
The concentratlon or amount of the antibactertal agent
absorbed Into each pad must glve an antlbacterlal sensltlvlty
correspondlng to that of the Klrby-Bauer method. The amount of the
antlbacterlal agent 1n the pad Is determlned by preparlng a serles of
pads eoch contolnlng the redox Indlcator and a dlfferent amount of the
antlbacterlal agent. The Inoculum of bacterla Is added to each pad
followed by Incub~tlon of thls serle~ of padfi. Eoch pad 1~ examlned
for the ~en~ltlvlty of the b~cterl~ to the concentratlon of the antl-
bacterlal agent, and from thls serles of concentratlons, the concentration
of the antlbacterl41 agent Is selected so thot the senstttvtty of the
bact~rla to thc antlbacterlal agent corresponds to the senslttvlty
exhlblted by the bacterla In the Klrby-i3auer method.
~ore speclf1cally, the amount of antlbacterla! agent In each
pad Is determlned by: ,
a) preparlng a plurallty of standard Inocula of bacteria,each
Inoculum betng of a dlfferent stroln of a parttcular bactertal
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specles and the selectTon of straTns TncludTng those
known to be sensTtTve and those known to be resTstant to the
antlbacterTal agent accordTng to the Klrby-Bauer method;
b) preparTng a correspondTng plurallty of a serTes of pads,
each pad Tmpregnated with the antTbacterTal agent 7n a different
concentratlon and a redox Tndicator Tn a concentration
selected so that the redox Indlcator changes color in response
to the growth of bacterta wlthln 3 to 36 hours at about 37C
when Inoculated wlth a predetermTned amount of saTd standard
Inoculum;
c) Inoculatlng eech serles of pads wlth the predetermined
~mount of a partlcular Inoculum of bactert fl;
d) Incubatlng the serles of pads at about 37C; and
e) selectlng the concentrotlon of the antlbacterlal agent
; 15 thot glves results equlvalent to those obtaTned by the KTrby-
Ebuer method
Not worthy Is the flndlng that the concentratlon of the
partlculor antlbocterlal agent, determlned In the precedlng manner, Is
sultoble for determlnlng the susceptlblllty of other bacterla to that
portlculor anttbocterlol agent, and con be employed as the standard
concentrotlon for that partlcular antlbacterlal ogent In the tray of
the opparatus The only llmltatlon to thls flndlng Ts~that dTfferent
concentrotlons ore requtrsd for gram neg~tlve m d gram posTtlve bacterTa
Any nutrlent broth Is sultoble as the medium for preparlng the
2S Inocull~m; for example, the broths d~scrlbed by W.R,BalIey and E G Scott
Dlognostlc Mlcrobiology The C.V. Mosby Company, Salnt Louls, U S A ,
1974, pp 363-388
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~ AHP-6781
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A convenient and preferred standardizatton of the inoculum
; of bacterta makes use of the brain heart infusion broth. In this
method the tops of isolated colonies, of a slmllar morphological type,
are transferred with a wire loop to 0.5 ml of brain heart Infusion broth
to glve a heavy suspensTon. The broth is incubated for thlrty
mlnutes at 37C. The turbidity is then adjusted with brain heart
Infuslon broth to a barlum sulfate standard correspondlng to a
MacFarland tube No. I or by adJustlng the suspenslon to an optical
denslty of 0.07 at 625 nm in a 13 mm dlameter test tube. Thls adjusted
suspenslon Is then dlluted 1/100 wlth braln heart Infuslon broth. Thls
dlluted suspenslon ~0.15 ml) Is odded to each of the 0.5 Inch dlameter
paper dlscs (descrlbed above) contalnlng the approprlate concentratlon
of Indlcator and antlbacterlal agent.
In applylng the method of thts InventTon for dstermlning the
susceptlblllty of bacterla to antlbacterlal agents equlvalent to the
standards of the accepted Klrby-eauer method, the followlng steps
are performed: applylng an Inoculum of bacterla to a pad, sald pad
conslstlng essentlally of an Inert mDterlal Impregnated wlth ~ redox
Indlcator and an antlbacterlal agent; Incub~tlng sald pad contalnlng
sald applled bacterla; and detectlng Dny change In sald redox Indlcator
as a mearure of the susceptlblllty of sald bacterla to sald antlbacterlal
agent; sald antlbacterlal agent, sald Inoculum of bacterla and sald
redox Indlcator belng so proportloned so that a color change Indlcates
reslstance of sald bacterla to sald antlbacterlal agent In accordance
wlth the results obtalned by the Klrby-eauer method.
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1~1214~
Other obJects and features of the Inventlon wlll become
apparent from conslderatlon of the followlng descr~ptlon of the preferred
embodlments taken In connectlon wlth the accompanying drawlngs.
Brl ~ e Drawlnqs
Flg. I Is a perspectlve vlew of an embodlment of the
apparatus accordlng to the present Inventlon;
Flg. 2 Is a cross-soctlonal vlew of the mbodlment of
Flg.l taken along the llne 2-2;
Flg. 3 15 a dotalled vl-w of a Jlngle Inert pad shown
Impregnat-d wlth a redcx Indtcator ~nd an antlbact-rlal agent; and
Flg. 4 Is a perspectlve vlow of another embodlment of an
apparatus accordlng to the present Inventlon.
pescrlDtlon of the Preferred Embodlm~nts
,
Referrlng now to the drawlngs and partlcularly to
, ~ F!gs. I and 2 ther- Is Illustrated an apparatus or device comprlsing
a rectangular tray 1~ and a complemental overlylng cover ll whlch 1~
plvotally mounted to the tray by hlnges 12 locatod at the bock of the
tray. Th- cov-r ~nd tr~y ~re ach formsd of a ultable pl~stlc
materlal preferably transparent and unaffocted by the materlals to
be placed In tho tray.
The tr~y 10 Is provlded wlth a plurallty of compartments 13
In the form of walls whlch can b- molded or oth-rwlse Integrally
formed ag part of the tray. Each compartm nt 13 contalns ~ pad 14 and each
pad 14 Is Impr-gnatod wlth the redox Indloator and an antlbacterla!
agent. Tho pad 14 Is advantageously slzed snugly to flt the compartment 13
so as to be securod thereln. the pad 14 can optionally b~ attached
to the bottom of the corpartment 13 by an adh~61ve.
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The front of the tray 10 and cover 11 is provided with
compl-mental snap fittings 15 whlch enable the cover and tray to be
snap-fitted or clamped together Tn tight fltting relation in order that
the pads 14 do not fall out of the compartment 13. The cover 11 or
tray 10 can be provlded wlth an Informatlon bearing marker ~not shown)
ad~iacent to each compartment 13 contalnlng the pad 14 for indicating
the type of antibacterial agent Impregnated In the pad 14.
One of the pads 14 can serve as a control. For lnstance, a
control pad can conta1n no antlbacterlal agent and the resultlng
color change wlll serve as an Indlcatlon of growth of the bacterla.
A socond control can conslst of a pad that contalns no antlbacterial agent
and remalns unlnoculated. It wtll show no color change.
Flg. 3 Illustrates the pad 14. Each pad 14 Is formed of an
Inert materlal, and Is Impregnated wlth a redox Indlcator that
15~ changes color In the presence of growlng bacterla, and In thls Instance
an antlblotlc of speelfled concentratlon.
Flg.4 Illustrates a perspectlve vlew of another embodiment
of an apparatus accordlng to the present Inventlon. In thts
embodlment the tray 10 Is fltted wlth a slldlng cover 16. Thc cover 16
slldes In th- guldes 17 whlch are formsd as ~ part of the tray 10.
The slldlng cov-r 16 may Include a handle 18 to provlde a handhold
for slldlng the cover l6. The tray 10 Is provlded wlth two parallel
row~ of comp~rtments 13, each contalnlng a pad 14 Impregnated with the
redox Indlcator and antlbacterlal agent. An extra compartment 19 Is provlded
In the tray 10, sald extra compartmont contalnlng ~ control pad 20
havlng the redox Indlcator but no antlbacterlal agent.
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~ AHP-6781
14a~1
In use, the apparatus constitutes an integrated package
which can readtiy be used by a technlclan. The cover 11 or 16 of the
apparatus of Flgure I or 4, respectively, Is opened so that the pads
14 In the various compartments 13 can be Tnoculated with an Tnoculum
of bacterla.
Next, the cover 11 or 16 Is closed and the apparatus is
Incubated at approxlmately 37C for 6 to 24 hours. Subsequent examination
of the color of the pads In the varlous compartments will indicate the
susceptlbillty or lack of susceptlbility of th0 organlsms to the
varlous antlbacterlal agents. Furthermore, the results wlll corre1ate
to those of the accopted Klrby-Bbuer method.
In a preferred embodlment of thls Inventlon we have selected
a preferred concentratlon of 3 ~9 of resazurln per 0.5 Inch dlameter
paper dlsc for gram posTtlve organlsms and 15 ~9 per 0.5 Inch diameter
paper dlsc for gram-negatlve organlsms.
Tho antlbacterlal agent llsted In the tables have the
followlng forms and potencles: amplclllln ttrlhydrate, 830 ~g/mg);
carbenlctllln tsodlum, 800 ~g/mg); cepholothln tsodlum, 945 ~g/mg):
chloramphenlcol (1,000 ~g/mg); cloxaclllln ~odlum, 910 ~g/mg);
erythromycln ~b~se, 910 ~g/mg); gontamlcln ~sulfate, 600 ~g/mg);
k~n~mycln ~sulfate, 805 ~g/mg); llncomycln ~hydrochlorlde monohydrate,
860 ~gtmg); neomycln (sulfato, 673 ~g/mg); penlclllln G tpotasslum,
1,580 unlts/mg), polymyxln B ~ulfate, 7,475 unlts/mg), streptomycln
; tsulfDte, 745 ~g/mg) and tetracycllne Ihydrochlorlde, 1,000 ~g/mg).
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AHP-678 1
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Table I gives the results of the combination of 3 ~9 per
dlsc of resazurtn and 0.0375 jug/dtsc of pentcllltn G when compared
to the Ktrby-8auer method agatnst six stratns of Sta~hylococcus
Dvo,qenes after 6 and 24 hours tncubation at 37C. In Table 1, and
the followtng tables, the pad employed Is the above descrlbed paper
dtsc No. 740-E.
~ AHP-6781
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TABLE I
Test Orqanism - StaDhvlococcus pyoqenes, Strains No. 12,40,41,126,127,128. .
Inoculum- Resazurin disc - 0.15 ml of standard7zed suspension is
~dded to the dlsc. --
KTrbv-Bauer - as outlined in the Federal Reglster, 37, (191),
20527, 1972.
Resylts - S - sensltive
R - resistant
R E S U L T S
~nKTrby-Bauer Resazurln
t24 hr) 6 hr 24 hr
,Y~ ~OCCUS
PYoaenes
No.12 S S S
R R R
41 5 S S
126 R R R
f 127 S S S
12~ 5 S S
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Table 2 gives the results of the combination of 15 ,ug of resazurin
per disc and 4.5 1l9 of ampîcillin when compared to the Kirby-Bauer method
against various species of gram negative bacteria after 6 and 24 hours
- incubation at 37C.
TABLE 2
Test Oraantsms: Proteus sD. No. 384; E. coli No. 119; ~E. coli No. 219
E. coli No. 253 Citrobacter sp. No. 305 Citrobacter sp.
No. 313 Klebsiella SD. No. 89, K!ebstella SD. No. 236.
Inoculum. - Resazurln dtscs - 0.15 ml of standardized inoculum
.~ Is ~dded to the dlsc.
Ktrb,~-E3auer - as outlIned In Federal Reglster, 37, (191),
.; 20527, 1972.
Results: S - sensltlve
R - reslstant
R E S U L T S
~peeles Klrbv-8~uer Rosazurlr~
t24 hr) 6 hr 24 hr
i'rote4s SD. No. 384 S S S
,~,. eoll No. 119 S ... ~ S S
- ~QLL No. 219 S S S
E. ~,L No. 253 S S S
Cltrob~cter sP. No. 305 R R R
Cltro_acter SD . No. 313 S S S
Klebslella sP. No. 89 R R R
Klebslella sP. No. 236 R R R
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AHP-6781
The results reported in Table I and Table 2 demonstrate
that the apparatus and method of this invention correlates at 6 hours
incubation with the Kirby-Bauer method with no subsequent change in
sensitivity if read after 24 hours incubation at 37C. This allows
the laboratory to obtain the results of the new sensitivity test of
this Inven;lon at any time between 6 and 24 hours incubation at 37C
knowing that the results will correlate with those obtained with the
Klrby-i3auer method after 24 hours Incubation.
For the selection of antibacterial agents concentrations
to be added to the pad in combination wlth the approprTate concentration
of resazurtn, the bacterla are dlvlded Into two groups, e.g., gram
posltlve and gram negatlve. In cllnlcal laboratorles perform7ng the
Klrby-Bauer sensltivity test, the choice of antlbacterial agents to
be us-d In the test Is based on whether the bacteria are gram positive
or gram negatlve. For the present Inventlon, the concentratlon of
antlbacterlal agents for the gram posltlve organisms 7s 17sted 7n
Table 3 and the concentrat70ns for the gram negat7ve bacter7a are
llsted In Table 4. Further examples of the correlatlon wlth the
Klrby-Ebuer m~thod for the grDm posltlve bacterla Is shown In Table 5
and for the gram negatlve bacterla, Table 6.
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TABLE 3
Preferred concentration of antibacterial agents (~g/disc) in combination
with 3 ~g/disc of resazurin for gram positive bacteria in general and
Enterococci in part1cular
J 5 Anttbacterial Agent Antibacterial Agent ; concentration (~g/dtsc) for concentration
AnttbacterTal Gram Postttve ~g/dtsc) for
Aaent _ 8acterla _ Entorococct
, . . -_ _ __
, Penicllltn G 0.0375 same
Cloxaclllln 1.5 same
Cephalothln 0.3 same
Chloramphenlcol 1.5 7.5
Erythromyctn 0.3 same
. Lincomycln 0.15 same
:~ Tetracycllne 1.5 same
Neomycln 1.5 same
Streptomycln 4.5 same
Amplclllln -* 1.5
* Note that sensltlvlty to penlclllln G Indlcates sensltlvlty to
amplclllln.
. -18-
-~~ AHP-6781
4~
- TABLE 4
Preferred concentration of antibacterial agents (~g/disc) in combination
with 15 ~g/dtsc of resazurin for gram negative bacteria
. Antlbacterial Agent
Antlbacterlal concentration
Agent ~g/disc) for
Gram Ne~ative ~acteria
Ampiclll1n 4~5
Cephalothln lO
Chloramphenlcol 7.5
10 Carbenlclllln 24
Gentamlcln 3
Kanamycln 4.5
Polymyxln B lOO
TetracyclIne 1.5
Neomycln 7.5
:' , :: ' :.
, ~, . :
AHP~
$~14~)
TABLE 5
.: Percent correlatlon of the resazurin-antibacterial agent disc method
~results read at 6 hours) to the Kirby-Bauer method against 79 clTntcal
isolates of StaDhy~occus pyoqenes.
Antlbacterial Aaent ~ Correlation
Penlcillln G 98.7
Cloxaclllln 10O
Cephalothin 100
Chloramphenlcol 100
*Erythromycln 100
Lincomycln 100
Tetracycllne 98.7
*After 18 hours Incubatlon.
-20- .
~~ AHP-6781
.
TAE~LE 6
Percent correlation of the resazurin-antibacterial agent disc method
(results read at 6 hours) to the Ktrby-Bauer method against gram
negatlve bacterla
, .
~ecle~_~nd ~ mfbers
E. coll - 20
Ente~obacter sp. - 15
Proteus ~ 6
Proteus vulqacl~ - 8
.Prote~la mlr3bl I ts - 10
~lebslella SD.- 12
Cltrobacter SD.- I
SeLcg~l~ SD-- 1O
Pseud,omQna~ aerualnos3 - 17
Total - 99 Gram negatlve bacterla.
: An ~ Aqent % Corre!atlon
Amplcllltn 92
*Cephalothln 95
*Chloramphenlcol 98
Gentamicln 99
; KDnamycln 99
*Polymyxln B 98
*Tetracycllne 99
, 25
f*18 hours Incubatlon necessary for: Cephalothln - E- co!l, E~tC9k~E~
Pro~ mlra,blll~si Chloramphenlcol - Proteus SD-, Klebsiella s~.;
Polymyxln B - ~err~tL~ SD-, ~S~y~ vu!~arls; Tetracycline - Serratia SD.
; ~ .
-21-
.
.
