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Agar well diffusion assay testing of bacterial
susceptibility to various antimicrobials in
concentrations non-toxic for human cells in culture
I. A. and S. T.
'Department of Molecular Genetics, Biochemistry and Microbiology and 'Department of Surgery, University of
Cincinnati College of Medicine and 'Shriners Bums Institute, Cincinnati, OH, USA
Previously, we'shaued that microbial susceptibility to antimicrobials testing was the recently described wet disc topical antimi-
in concentrations non-toxic for human celk in culture could be tested crobial assay (WDA)'O.". However, an agar well diffusion
hst described by Nathan et al. in 1978, has
wing the wet disc topical antimicrobial assay. In this report, wet disc assay (AWDA)
assay and agar well difiion assay results were compared testing the been the model most commonly used by bum units for
susceptibility of Ps, aeruginosa isolates from bum patients to testing topical antimicrobial activity"-".
concentrations of Polymyrin B non-toric for cultured cells. Both assays Therefore, using antimicrobial concentrations non-toxic
perfonncd on the same agar plates. No differences in results were for human cells in culture, we compared these two methods
were to determine if the AWDA results were similar to results
obserued. Further agar well diffusion assay testing showed that using the WDA. Susceptibility of Ps. aeruginosa isolates to
susceptibilitylresistance could be demonstrated when testing several the antimiaobial drug Polyrnyxin B was used for this
antimicrobials In concentrations non-toxic for cultured cells against a comparison. After it was determined that results using the
oariety of bacteria isolated from bum patients. Therefore, the more two methods were comparable, additional bacterial iso-
familiar agar well diffusion as well as the wet disc assay can be used to lates were tested in the AWDA for susceptibility to other
test microbial susceptibility to these concentrations of antimicrobials. antimicrobials, alone or in combination. Results of these
Bums (1994) 20, (5). 426429 two studies are presented in this report.
Introduction Materials and methods
Bacteria
Cultured skin grafts have become a potentially important Fresh Gram-negative bacteria and Staph. aureus isolated
therapy for the closure of bum wounds, chronic ulcers and from the wounds of patients at this institution were tested.
sites of reconshuctive surgery1. All cultured sh graft
models contain keratinocytes, and some also contain Antimicrobials
biopolymer implant. All are avascular and
fibroblasts and a Mupirocin (MUP) and Sparfloxacin (SPAR) powders were
rnicro- supplied through the generosity of Smith Kline Beecham
partially keratinized, and therefore easily subject to
bial destruction'-'. Because of these biological deficiencies Pharmaceuticals (Philadelphia, PA, USA) and Parke-Davis
topical antimiaobial treatment used in conjunction with Pharmaceutical Research Division (Ann Arbor, MI, USA),
cultured skin grafts is necessary. A number of parenteral respectively. Polymyxin B (POLY B: 500 000 unit vials;
and topical antimicrobial agents are toxic for cultured cells, Rorig/P&er & Co., New York, USA) was supplied by our
and because of this are excluded from Conversely, a pharmacy. All antirniaobial solutions were prepared in our
number of both parenteral and topical antimicrobial solu- hospital pharmacy using sterile saline as the diluent for
B, 40 mg, was dissolved in I ml of
in certain concentrations not to be MUP and SPAR. POLY
tions have been shown
toxic for cultured cells'. While this report suggested that polyethylene glycol before being diluted to 100 ml with
those concentrations of antimicrobials could be used sterile saline. After warming of the solution to effect
clinically, no demonstration of retention of antirniaobial complete solubilization of the POLY B, appropriate
activity was shown for the solutions at those concentra- dilutions were prepared in sterile saline.
tions. After testing in our recently described cellular cytotoxi-
Recent publications from this institution have addressed city assay8, it was found that 20 pg/ml SPAR, 400 U/ml
both of these issues and presented methods to determine POLY B and 32 pg/ml MUP caused no toxicity to either
concentrations of antimicrobial drugs non-toxic for cells in keratinocytes or fibroblasts9. These concentrations were
culture and to test whether these drug concentrations used in susceptibility test assays in vitro to determine their
retained antimiaobial a~tivity',~. activity against bacteria isolated from our patients. While
In our studies the method that we used for antimicrobial SPAR has a spectrum of activity against both Gram-
0 1994 Butteworth-Heinemann Ltd
0305-4179/94/050426-04
Holder and Boyce: Bacterial susceptibility to antimicrobials
negative and Gram-positive bacteriaL6 it was tested only culturela. Results using mixtures were compared to results
against Gram-negative bacteria
in this study. using individual antimicrobial solutions.
Antimicrobial susceptibility test methods Stability study
Initially, susceptibility of Ps. aeruginosa isolates to POLY B Because testing was projected to require several weeks, the
was tested using both the WDAIO," and a modification of stability of the antimicrobial solutions over time was
the AWDA1', to determine if the results using the two determined. Prior to routine testing, SPAR and POLY B
procedures were comparable. For both methods, the test were tested for 4 consecutive weeks using thg WDA,
microorganism, grown up to a density of an 0.5 Mac- against the same two susceptible isolates of Ps. aeruginosa;
Farlane Standard in brain-heart infusion broth, was poured MUP was tested against the same two susceptible isolates
mm of Staph. aurew. The antimicrobial solutions were refnger-
evenly over the surface of commercially available 150 ated between tests. Stability was determined by comparing
Mueller-Hinton agar plates (BBL; Cockeysville, MD, the zone diameters after each test. If the zone diameter was
USA). After the excess inoculum was decanted, the plate the same as the initial zone diameter f I mm, the solution
surface was dried by placing the plate in an incubator was considered stable for that time period. Using this
(35°C) for 10 min. For the WDA sterile filter paper discs (6 criterion, SPAR was stable only for 3 weeks. Therefore.
mm) wetted with 25 p1 of antimicrobial solution were &week basis.
placed on the surface of the inoculated plate. fresh solutions for testing were prepared on a
-For the AWDA. a modification of the published method Statistical analysis
originally designed to test the efficacy of topical antimicro- Results of the WDA versus AWDA dose response were
bial creams and ointments was used1'. Plates inoculated compared using a one-between, one-within repeated
with the test organism had 6-mm wells cut into the surface measurements of analysis of variance. Differences were
of the agar using a cork borer dipped in alcohol and flamed. considered significant if P< 0.05.
The wells were filled with 100 p1 of antimicrobial solutions.
All plates were incubated (35°C) overnight. Results
After incubation, the diameters of any clear zones Dose response testing of POLY B for its antimicrobial
around the antimicrobial-containing discs or wells were activity against dinical isolates of Ps. aacgirfosa was
measured using calipers. Because the antimicrobial agents compared using WDA and AWDA techniques on the same
would be used prospectively as wet soak dressings directly test plates (Table 0. With 10 U POLY B absorbed into the
over the cultured skin grafts, and therefore would be in discs or placed in the wells, only 50-60 per cent of the
direct contact with the bacteria colonizing the surface of isolates tested were susceptible. In contrast, all
graft or graft bed, it was decided that a zone of dearing Ps. am-
the ginow strains tested were susceptible when 20-40 U of
around the test disc or agar weU of 2 1 mm in radius (i.e. a POLY B were placed on discs or into wells. No significant
total zone diameter measurement of 28 mm) would be differences in mean zone diameters were seen after any
taken as susceptibility of the test bacterial strain to the equimolar weight of POLY B was compared using the
antimicrobial. WDA versus the AWDA.
Experimental studies This comparison showed that results obtained using the
Initially, a dose response experiment comparing the AWDA were the same as results obtained using the WDA.
activities of various amounts of POLY B against 12 dinical Therefore, additional testing of antimicrobials (MUP,
isolates of Ps. aeruginosa was performed using both the SPAR, POLY B) against a variety of clinical isolates of
WDA and the AWDA. 100-p1 aliquots of POLY B were bacteria was conducted using the AWDA. Results of this
delivered to sterile 6-mm filter paper discs in a volume of testing are presented in Table 11. Except for POLY B, alone
25 pl. Each disc was placed on the same plate, close to the or in combination with MLJP tested against Protacs
well containing the comparable amount of POLY mirabilis strains and against one strain of Serratia mnrce-
B, but far Kens, all bacteria were susceptible to the antimicrobial
enough away that any zones of inhibition could be dearly tested. Comparisons of single antimicrobial agents yersus
read and measured. This experiment allowed us, simultan- antagdnistic
eously, to determine the optimal concentration of antimi- mixtures showed no additive, synergistic nor
crobial solution to use for testing and to determine if actions. POLY B was totally inactive against P. mirabilis,
- results using AWDA were comparable to results obtained and no change occurred' when POLY B:MUP mixtures
using the WDA. were tested. Gram-negative bacteria were uniformly sus-
After it was determined that results using the AWDA ceptible to SPAR with or without the addition of MUP and
were equivalent to those obtained using the WA, an all Staph. aurw isolates tested susceptible to MUP with or
expanded study was performed using the AWDA to test without SPAR or POLY B.
the efficacy of SPAR, POLY B and MUP against a wide
variety of clinical isolates of bacteria obtained from bum Table I. Comparative results of wet disc assay (WDA) and agar
patients. One hundred microlitre volumes of the concen- well diffusion assay (AWDA) testing: Polymyxin B versus
trations of these antimicrobials found to be non-toxic for P. aeruginosa tested on the same plate
cells in culture were used.
In addition, to determine whether mixtures of these POL Y B (U) tested WDA A WDA
antimicrobials would be synergistic, additive, antagonistic 10 8.9i 4.3 (7)' 12.2 i 1 .O (5)
or neutral to each other, combinations of both SPAR and 20 10.4
POLY B with MUP were prepared to contain concentra- i 2.4 (1 2) 11.2i2.4 (12)
30 12.4
tions of antimicrobial agents that were isomolar to the i 2.2 (1 2) 11.2i5.0(12)
individual preparations. These antimicrobial mixtures have 40 12.9i 1.8 (12) 13.2 i3.2 (1 2)
also been shown to be non-toxic for human cells in 'Mean zone diametef i s.d. (no. susceptible out of 12).
428 Burns (1994) Vol. 20/No. 5
Table II. Agar well diffusion assay results testing susceptibility of bacteria isolated from bum patients to Sparfloxacin', Polymyxin B*
and Mupirocin', alone and in combination
Ps. Enrerobacrer Klebsiella Escherichia Proreus Serrarfa Acinetobacter Staph.
aeruginosa cloacae pneumoniae coli mirabilis marcescens baumannii aureus
Antimicrobial (n = 33) (n = 19) (n= 19) (n=9) (n=8) (n=10) (n = 7) (n = 33)
MUP NT NT NT NT NT NT NT 27.1 * 1.5
SPAR 20.0*4.3' 25.9i 2.0 24.9* 2.2 30.9f 1.7 24.3f 5.9 20.7f 3.0 28.4i 3.3 NT
SPAR:MUP 19.8*4.0 26.3*2.0 24.4f 2.3 31.3il.O 24.0f 4.0 21.1
POLY B 12.4*0.9 10.7f0.7 10.9f 1.1 11.8i 0.4 0 *3.2 28.1 f3.0 26.3+2.0
POLY B:MUP 12.3i1.0 11.2f0.7 11.6i 1.4 11.0f0.3 0 12.0f1.3' 12.9f1.7 NT
12.0* 1.5: 13.1 3~2.1 26.8+1.9
'1 00p1 of concentration. in either pglml or Utml, found to be non-toxic for cells in culture per well.
NT, not tested.
:Mean zone diameter*s.d. in millimetres.
.One isolate= no zone.
Discussion Acknowledgements
Our results demonstrate that both the WDA and the The authors thank Paula Durkee, Margie Hartzel and Jim
AWDA were equally valid for antimicrobial testing (Table Wesselman for their technical support on this project.
0. The antimicrobial activity of POLY B was concentration
dependent in both assays, since discs or wells containing
only 10 U of POLY B inhibited the growth of only five to
seven strains of Ps. aeruginosa, whereas discs or wells References
containing 220 U POLY B uniformly inhibited all 12
strains tested (Table 0. Why seven of 12 strains tested 1 Carver N and Leigh IM. Keratinocyte grafts and skin
susceptible in the AWDA, is not clear. Perhaps the kinetics equivalents. Int] Dmtol 1991; 30: 540-541.
of diffusion of the antimicrobial agents into the agar from a 2 Gallico 111 GG, O'Conner NE, Compton CC et al. Permanent
surface application (WDA) is different from diffusion coverage of large bum wounds with autologous cultured
through the agar (AWDA) when the antimiaobial is Nm Engl] Med 1984: 311: 446-451.
human epithelium.
placed into a well.
Alternatively, the fact that the antimi- 3 Hansbrough JF, Boyce ST, Cooper ML et al. Bum wound
crobial solution was applied to discs in only 25 p1 amounts closure with cultured human keratinocytes and fibroblasts
attached to a collagen-GAG subshate.
while 100 pl of solution was placed in wells, might account JAMA 1989: 262:
for the difference. In any case, these results demonstrate 2125-2130.
that both assays discriminate between susceptible and 4 Hull BE, Finley RK and Miller SF. Coverage of bums with
resistant strains. When higher amounts of POLY B were bi-layered skin equivalents: a preliminary clinical trial.
used in either the disc or well format, all isolates were Surgery 1990; 107: 496-502.
susceptible. 5 Cooper ML, Boyce ST, Hansbrough JF et al. Cytotoxiaty to
Susceptible versus resistant strains of bacteria were cultured human keratinocytes (HK) of topical antimiao-
apparent using the AWDA even when 100 p1 of antimi- bial agents. ] Surg Res 1990; 48: 190-195.
crobial agents in concentrations found non-toxic for cells 6 Lineweaver W, McMorris S, Soucy D et al. Cellular and
in culture were placed in wells (Table 0. This is illustrated bacterial toxicities of topical antimicrobials. Plast Recomb
by comparing the 100 per cent susceptibility of P. mirabilis Surg 1985; 75: 394-396.
strains to SPAR, alone or mixed with MUP, with the total 7 Barillo DJ, Nangle ME and Farrel K. Preliminary experience
resistance of these same strains to POLY B or POLY with cultured epidermal autograft in a community hospital
B:MUP. In addition. one strain of S. mnrcescens was bum unit. ] Bum Care Rehubil1992; 13: 158-165.
resistant to both POLY B and POLY B:MUP while nine 8 Boyce ST and Holder Ik Selection of topical antimiaobial
others were susceptible. In all other cases each antimicro- agents for cultured skin for bums by combined assessment
bial agent inhibited tlie in vitro growth of appropriate of cellular cytotoxiaty and antimiaobial activity. Plast
Gram-positive or Gram-negative bacteria enough to be Recomb Surg 1993; 92: 493-500.
considered effective against those organisms by the 9 Boyce ST and Holder IA. Cytotoxiaty testing of human
criteria set up in this study. keratinocytes and fibroblasts to topical antimicrobial
In summary, the data show that determinations of agents for use with cultured slun grafts. Proc Am Bum
bacterial susceptibility/resistmce to antimicrobial agents AsXK 1993; absh 7.
in concentrations non-toxic for cells in culture are directly 10 Holder IA. The wet disc antimiaobial solution assay: an in
comparable using the WDA or the AWDA. Furthermore, viho method to test efficacy of antimicrobial solutions for
100 pl of these concentrations of antimicrobial agents topical use. ] Bum Care RPhabil 1989; 10: 203-208.
placed in the well in the AWDA discriminates between II Holder IA. Wet disc testing of mafenide hydrochloride,
resistant and susceptible bacteria. chlorhexidine gluconate, and triple antibiotic solution
We conclude, therefore, that in addition to the WDA, the against bacteria isolated from bum wounds. I Burn Carc
more familiar AWDA can be used to test susceptibility/ Rehabil1990; 11: 301-304.
resistance of microorganisms to concentrations of antimi- 12 Heggen JP, Velanovich V, Robson MC et al. Control of bum
crobial agents non-toxic for cells in culture. wound sepsis: a comparison of in vitro topical antimiao-
Holder and Boyce: Bacterial susceptibility to antimicrobials 429
bial assays. Bum Care Rehabil 1987; 8: 176-179. quinolone with high activity against gram-positive bac-
13 Holder IA, Schwab M and Jackson L. Eighteen months of teria. Diag Mimobiol Infect Dis 1991; 14: 403-415.
routine topical antimicrobial susceptibility testing of 17 Nathan P, Law El, Murphy DF &a]. A laboratory method for
isolates from bum patients: results' and conclusions. ] selection of topical antimicrobial agents to treat infected
Antimimob Chemother 1979; 5: 455-463. bum wounds. Burns 1978; 4: 177-187.
14 Rode H, Hans10 D, deWet PM et al. Efficacy of mupirocin in 18 Boyce ST, Warden GD and volder IA. Non-cytotoxic
methicillin-resistant Staphylococcus auracs bum wound combinations of topical antimicrobial agents for use with
infection. Antimimob Apenfs Chemother 1989; 33: 1358- cultured skin. i+oc Am Bum Assoc 1994; (abstr. 103).
1361. -
15 Thompson PD, Taddonio TE, Tait MJ et al. Susceptibility of Paper accepted 2 February 1994.
Pseudomonas and Staphylococcus wound isolates to
topical antimicrobial agents: a 10-year review and clinical
evaluation. Burns 1989: 15: 190-192. Correspondmc~ should bc addressed to: Dr I. A. Holder, Shriners
16 Cohen MA, Huband MD, Mailloux GB et al. In vitro activity Bums Institute, 3229 Bumet Avenue, Cinannati, OH 45229.
of Sparfloxaan (CI-978, AT-4140, and PD 131501) a USA.
~TH ~NTERNATIONAL SEMINAR ON WOUND
HEALING AND WOUND MOVEMENT
CHICAGO, ILLINOIS, USA
OCTOBER &9,1994
The International Bum Foundation is sponsoring the 7th annual two day International
Symposium on Wound Healing & Wound Management, October 8-9, 1994 in Chicago. This
is a Saturday and Sunday preceding the Clinical Congress of the American CoUege of
Surgeons.
This meeting is designed to assemble some internationally mogmd experts in wound healing
and wound management to discuss the state of the art in these topics. The indepth program
will cover basic and clinical research and application to patient care.
Potential topics incluk
principles of wound healing growth factors
synthetic skin biosynthetic dressings
epithelial
cell cultures cultured dermis and epidermal tissues
wound assessment immune response & wound healing
wound hormones prostaglandin's in wound healing
pressure sores wound contracturn
For further information please contact: Dr John A. Boswick, FACS., Course Director,
International Bum Foundation, ZOOS Franklin Street, #355, Denver, Colorado 80205, USA.
Tel: (303) 839 1694 or Fax: (303) 839 1695.
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