The Effect of Brief Exposure to Sub-Therapeutic Concentrations of Chlorhexidine Digluconate on the Susceptibility of Staphylococci to Platelet Microbicidal Protein

Patients and Methods

Bacillus subtilis ATCC 6633 (American Type Culture Collection, Rockville, MD), S. aureus ATCC 6538P, and S. epidermidis ATCC 14990 are well-characterized laboratory strains. Unless otherwise stated, chemicals were obtained from Sigma Chemical Corporation (St. Louis, MO). Formulated bacteriologic media were purchased from Difco Laboratories (Detroit, MI).

Human PMP was prepared and standardized as described earlier [9]. The bioactivity of PMP against B. subtilis ranged from 0.5 to 1.0 mcg/mL. Well-characterized clinical isolates of S. aureus (n=15) and CoNS (n=10) were from Collection of Microorganisms (Institute of Cellular and Intracellular Symbiosis, Russian Academy of Sciences, Orenburg, Russia) and used for clarity of experiments.

Susceptibility of S. aureus and CoNS to the bactericidal action of PMP was tested as described previously [9]. On the basis of our original findings [19], we designated a breakpoint for staphylococcal susceptibility to PMP as the minimal concentration of protein at which 20% or more inhibition of bacterial growth was determined. To define the proportion of strains considered PMP-susceptible or PMP-resistant, PMP concentration of 5 mcg/mL was considered to be a relative PMP-susceptibility breakpoint, as in the data of Wu et al. [10].

The influence of brief exposure to three different sub-therapeutic concentrations of CHXD (0.005%, 0.0025%, and 0.00125%) on the subsequent staphylocidal effect of PMP was evaluated as recommended by Ellepola and Samaranayake [17]. Sub-therapeutic concentrations of CHXD were obtained from serial dilutions of medicinal preparations of CHXD (0.05% solution) kindly provided by Lekar LTD (Moscow, Russia). Chlorhexidine digluconate pre-treatment was performed with an initial bacterial inoculum of 10⁹ colony forming units per milliliter (CFU/mL) in phosphate-buffered saline (PBS) for 1 h at 37°C with agitation. Chlorhexidine digluconate pre-treated or control bacterial cells were harvested in parallel by centrifugation at 3,000× g for 15 min, washed twice in PBS to remove residual CHXD, and re-suspended in PBS to final inoculum of 10⁹ CFU/mL. A 100-mcL volume of each final bacterial inoculum was added to tubes containing 900-mcL PMP dilution or to control tubes containing PBS only. All assays were conducted more than 1-h incubation period at 37°C. Aliquots at the end of the incubation period were plated on blood agar plates. Colonies were counted after overnight incubation at 37°C and the number of surviving organisms were calculated. Percentage of growth inhibition was calculated using the formula: percentage inhibition=(N_k − N_o)/(N_k)×100, where N_k was the number of surviving staphylococcal cells in the absence of CHXD pretreatment and N_o was the number of surviving staphylococcal cells after the CHXD pre-treatment. The final data are mean±standard deviation (SD). The differences in bactericidal rates between S. aureus or CoNS cells exposed to PMP in the presence or absence of CHXD pre-treatment were compared by the unpaired Student t test. A p value of ≤0.01 was considered to represent a significant difference.

Results

Among all clinical staphylococcal strains studied, all isolates were considered to be resistant to the bactericidal action of PMP (Table 1). Moreover, 9 of 15 strains of S. aureus (60%) were highly resistant to of PMP (<15 mcg/mL). On the other hand, of the 10 CoNS isolates tested, only 20% (p<0.001) were highly resistant to PMP. Platelet microbicidal proteins bioactivity against reference strains S. aureus ATCC 6538P and S. epidermidis ATCC 14990 ranged between 3.0–5.0 mcg/mL and 2.5–4.0 mcg/mL, respectively. On the basis of our findings, the final PMP concentration of 10 mcg/mL was used in all subsequent studies.

Exposure of S. aureus or CoNS cells to CHXD prior to PMP resulted in significantly increased staphylococcal killing compared with the killing achieved with PMP alone (control cells) (Table 2). This enhanced effect was most marked for concentrations of CHXD of 0.005%.

Discussion

Endovascular infection is a serious complications, frequently resulting in prolonged hospitalization, organ failure, and death [1,5]. Thus, antiseptic agents are widely used in hospitals and are essential when prevention and control of nosocomial infections are required. Chlorhexidine was first described in 1954. The primary mechanisms of action of this biocide is membrane disruption, causing growth inhibition and cell death [14]. Maisetta et al. [20] showed that chlorhexidine might facilitate the access of human β-defensin 3 to the cytoplasmic membrane via damaging the outer bacterial membrane, which represents the main site of action. Similarly, Koontongkaew and Jitpukdeebodintra [21] found the effect of CHXD on cell membranes of Streptococcus mutans GS-5 after treating the extracted proteins for 30 min with the drug at final concentrations of 0.05% and 0.2% chlorhexidine caused selective reduction in the intensity of the membrane proteins. In addition, Kim et al. [22] showed marked synergistic anti-bacterial effects of gaegurin 6 (GGN6), an animal-derived cationic peptide, and its derivatives PTP6 and PTP12 with chlorhexidine on the growth of oral streptococci.

Collectively, our results and many other reports have demonstrated that the site for anti-bacterial action of PMPs is the cytoplasmic membrane, where they cause the formation of voltage-gated channels that span membranes without requiring a specific target receptor [8]. On the other hand, Kupferwasser et al. [23] demonstrated that the staphylococcal multi-drug-resistant gene qacA mediated resistance to PMP in vitro. Moreover, it is shown that reduced biocide susceptibility in staphylococci is associated with quaternary ammonium compound (qac) gene-encoding efflux proteins [24]. We suggest that reduced anti-septic and PMP susceptibility may allow persistence of organisms in the presence of low level residues and contribute to survival of staphylococci. Taken together, our findings suggest that short exposure to subtherapeutic levels of CHXD may enhance staphylococcal susceptibility to mammalian cationic peptides, thereby suppressing its pathogenicity in vivo. The synergistic anti-staphylococcal properties of PMP and CHXD demonstrated in the present study suggest that PMP has an important in vivo role in the defense against vascular infections. In summary, PMP exerts cooperative bactericidal effect with anti-septics. These observations provide an in vitro basis for the synergistic role of platelet host defenses in the anti-septic prophylaxis of blood stream staphylococcal infections [17]. Collectively, these results provide evidence that platelets play important roles in host defense against endovascular infection and that these effects may be amplified in the presence of anti-septics [7,8]. Moreover, the recent study by Karpanen et al. [25] showed that the CHXD intravascular catheter site gel dressing should suppress bacterial growth on the skin at the catheter insertion site, thereby reducing the risk of infection. On the other hand, the absence of dose-dependent susceptibility of CNS to PMP is interesting. We believe that it may be explained by the presence of novel regulatory system with a yet unknown function encoded in the genome of S. epidermidis next to the defensin-regulated vraFG-homologous transporter genes [26]. Moreover, Li et al. [26] found that genome of S. epidermidis contains a classic two-component signal transducer and an unusual third protein, all of which are indispensable for signal transduction and anti-microbial peptide resistance.

In a broader sense, the present findings support a significant and beneficial interaction between innate immune mechanisms and exogenous anti-infective agents, yielding a net synergistic impact. From these perspectives, our present data show that platelets contribute significantly to anti-microbial host defense and potentiate the anti-microbial mechanisms of distinct classes of conventional anti-staphylococcal agents. These results support the hypothesis that exogenous antimicrobial agents and endogenous mechanisms of antimicrobial host defense interact to yield mutual potentiation resulting in amplified anti-microbial efficacy. Thus, the anti-staphylococcal PMP and CHXD synergistic activity in vitro demonstrated in present study make these molecules potentially useful for prevent endovascular catheter-associated infections. The combined data indicate that PMP exerts cooperative bactericidal effect with CHXD. The anti-staphylococcal PMP and CHXD synergistic activity in vitro demonstrated in the present study make these molecules potentially useful for preventing endovascular catheter-associated infections. The synergistic, anti-staphylococcal in vitro activity of the CHXD/PMP combination and the future research of the in vivo efficacy of catheters coated with this unique composition encourage clinical evaluation of this innovative approach. This future research based on animal and human models is needed to elucidate their in vivo efficacies and toxicities and their utility in clinical practice.