Keaton Smith1, M.S.; Abteen Moshref1, B.S.; Amber Jennings1, Ph.D.; Harry Courtney2, Ph.D.; Warren Haggard1, Ph.D. 1The University of Memphis Department of Biomedical Engineering

2The University of Tennessee for Health Sciences Department of Medicine

Table 1. MIC, FICI and the resulting effect for various antimicrobials against S. aureus.Antimicrobial MIC (µg/ml) FICI Effect

Amikacin (AMK) 0.5 - -

Daptomycin (DAP) 0.5 - -

Vancomycin (VAN) 0.5 - -

Chlorhexidine (CHX) 0.5 - -

CHX + AMK - 0.750 Additivity

CHX + DAP - 1.500 Additivity

CHX + VAN - 0.375 SynergismFICI ≤ 0.5 indicates synergy, 0.5 < FICI < 2.0 indicates additivity, 2.0 ≤ FICI < 4.0 indicates indifference and FICI ≥ 4.0 indicates antagonism (n = 1).

Table 2. The average activity for eluate samples against S. aureus.Antimicrobial Time (days)

1 2 5 10 14 21

VAN ‒ ‒ + + + +

CHX ‒ ‒ ‒ ‒ ‒ ‒

CHX + VAN ‒ ‒ ‒ ‒ ‒ ‒"‒" indicates S. aureus inhibition and "+" indicates S. aureus growth (n = 5).

1 2 5 10 14 211

10

100

1000

VAN CHX

VAN from Combo CHX from Combo

Time (days)

Co

nc

en

tra

tio

n (

µg

/ml)

0.49 0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 1000.000

20

40

60

80

100

120

VAN CHX

CHX + VAN

Concentration (µg/ml)

Pe

rce

nt

Ce

ll V

iab

ility

Figure 1. Cell Viability

Figure 2. Antimicrobial Release Profile

Antimicrobial Combination Exploits Synergismand Local Drug Delivery for Infection Therapy

IntroductionInfections in military and civilian populations

continue to be serious problems for patients with contaminated traumatic wounds as well as with infected surgical sites. An additional complication is the increase in bacterial resistance towards antibiotics [1]. With few new antibiotics being developed and marketed, there is a need for novel treatment methods utilizing currently available antimicrobial agents [2].

In this investigation, we aim to exploit the beneficial pharmacodynamic effects between multiple antimicrobials in combination with local drug delivery via a previously established drug delivery device [3,4]. The successful development of this treatment method could be applied towards chronic clinical infections, offering the more effective use of a lower antimicrobial agent dose.

MethodsAntimicrobial Synergism. The minimum inhibitory concentration (MIC) was determined for each antimicrobial tested via the turbidity of S. aureus treated at varying antimicrobial concentrations. A checkerboard method was used to combine two antimicrobials at various concentrations to determine the MIC of each antimicrobial in each combination [5]. The fractional inhibitory concentration index (FICI) was determined by the following equation: (Drug A MIC combined / Drug A MIC alone) + (Drug B MIC combined / Drug B MIC alone). The value of the resulting ratio indicated the pharmacodynamic effect (see Table 1).

Cell Viability. NIH/3T3 fibroblast cell viability was assessed using Cell Titer–Glo Luminescent Cell Viability Assay (Promega Corp., Madison, WI). This assay determined the number of viable cells after treatment with standard concentrations of antimicrobials, based on quantification of the ATP, which signals the presence of metabolically active cells (Figure 1, n = 3).

Antimicrobial Release. The elution profile of antimicrobials released from a chitosan sponge local delivery system was determined at various time points and analyzed using HPLC (Figure 2, n = 5). All eluate samples were subjected to antimicrobial activity testing against S. aureus via turbidity analysis (Table 2, n = 5).

Statistical Analysis. Statistical differences between cell viability and antibiotic release were determined using two-way ANOVA with Tukey’s post-hoc analysis and α = 0.05 (Sigmaplot v3.5, San Jose, CA).

Results► The combination of chlorhexidine and vancomycin

resulted in pharmacodynamic synergism against the S. aureus strain tested (Table 1).

► Alone, chlorhexidine significantly decreased cell viability at concentrations ≥ 3.91 µg/ml (p < 0.001) and in combination with vancomycin at concentrations ≥ 7.81 µg/ml (p ≤ 0.042) (Figure 1).

► Chlorhexidine and vancomycin were released in a bolus individually and in combination, and their release profile was statistically similar after the day 5 time point (Figure 2).

► All eluent samples were active except for those whose concentrations approached the MIC after the dilution procedures.

Discussion/Conclusion► Our investigation found one combination of

antimicrobials with synergism which may be delivered via a delivery device to a local environment, inhibiting the growth of S. aureus.

► Although chlorhexidine may decrease fibroblast activity, its transient presence in a local infected wound environment may not inhibit wound healing. No other studies could be found investigating the local delivery of these selected antimicrobials. However, other antimicrobials are being investigated in vitro, for their systemic, synergistic effects against infectious microbes [6-8].

► With further in vivo research, the application of synergistic, vancomcyin and chlorhexidine antimicrobials, via a local drug delivery device could provide a more efficient treatment for bacterial infections.

► Additional research should continue in order to identify synergism between additional antimicrobial agents and alternative local delivery methods, which could prove beneficial towards infection therapy.

References1. Guillemot D. Antibiotic use in humans and bacterial resistance. Curr Opin Microbiol. Oct

1999;2(5):494-498.2. Chan M. Antimicrobial resistance in the European Union and the world. Conference on

Combating Antimicrobial Resistance. Copenhagen, Denmark: World Health Organization; 2012.3. Noel SP, Courtney HS, Bumgardner JD, Haggard WO. Chitosan sponges to locally deliver

amikacin and vancomycin: a pilot in vitro evaluation. Clin Orthop Relat Res. Aug 2010;468(8):2074-2080.

4. Stinner DJ, Noel SP, Haggard WO, Watson JT, Wenke JC. Local antibiotic delivery using tailorable chitosan sponges: the future of infection control? J Orthop Trauma. Sep 2010;24(9):592-597.

5. Lorian V, ed Antibiotics in Laboratory Medicine. 5 ed. Philadelphia: Lippincott Williams & Wilkins; 2005.

6. Silva LV, Araujo MT, Santos KR, Nunes AP. Evaluation of the synergistic potential of vancomycin combined with other antimicrobial agents against methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp strains. Mem Inst Oswaldo Cruz. Feb 2011;106(1):44-50.

7. Araoka H. [Combination therapy against multidrug-resistant bacteria]. Nihon Rinsho. Feb 2012;70(2):305-310.

8. Dosler S, Gerceker AA. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Chemotherapy. 2011;57(6):511-516.