Evaluation of the effects of chlorhexidine digluconate with and without cBD103 or cCath against multidrug-resistant clinical isolates of Staphylococcus pseudintermedius.

BACKGROUND: Because of the increased incidence of multidrug-resistant (MDR) bacteria, the use of disinfectants over antibiotics has been encouraged. However, the interactions between disinfectants and host local immunity are poorly understood.
OBJECTIVE: To assess the effects of chlorhexidine digluconate (Chx), with and without selected host defence peptides (HDPs), against MDR Staphylococcus pseudintermedius (MDR-SP). METHODS AND MATERIALS: Ten clinical isolates of MDR-SP were tested, using a modified microbroth dilution method. Four two-fold dilutions of 2% Chx and 1 μg/mL the HDPs synthetic canine β-defensin 103 (cBD103) or cathelicidin (cCath) were tested alone or in combination. Colony counts after 5, 15, 30 and 60 min, and a minimum inhibitory concentration (MIC) after 24 h were recorded. Friedman followed by Dunn's multiple comparison tests with significance of P < 0.05 were used for statistical analysis. Synergy, additivity/neutrality or antagonism were calculated.
RESULTS: Growth was not inhibited by either HDP alone. An MIC of 0.312 μg/mL Chx was achieved for nine of the isolates. One isolate had an MIC of 0.078 μg/mL Chx. A MIC90 (in nine of 10 isolates) of 0.312 µg/mL was seen for Chx in combination with either HDP. Synergy was seen in the combination Chx/cCath used at the highest concentrations of Chx (0.624 µg/mL and 0.312 µg/mL) after 30 and 60 min incubation. Additivity/neutrality was seen for most of the other concentrations and times of incubation. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest a synergistic/additive effect between Chx and HDPs in dogs. Further studies evaluating the mechanisms behind this effect are needed.

Introduction

The incidence of antimicrobial resistance has increased over the past few years, with multidrug‐resistant (MDR) organisms being commonly isolated in clinical practice. , In particular, the isolation of MDR Staphylococcus pseudintermedius (SP) is becoming a common finding in veterinary dermatological practice. ,

Unfortunately, the rate of discovery of new antibiotics has plateaued, leaving – in some cases – topical antimicrobials as the only viable option to treat such infections. Among others, chlorhexidine digluconate (Chx), a biguanide compound, has been used successfully as antimicrobial agent against many Gram‐positive and Gram‐negative bacteria, yeasts, moulds and viruses. , Its mechanism of action (MoA) is not completely understood, yet it seems related to its ability to bind and interfere with bacterial membranes. This strong binding induces structural modifications leading to a leakage of the intracellular components. In veterinary medicine, Chx is used widely in clinical dermatological practice (in concentrations between 0.5% and 4%) as sole or adjuvant therapy for cutaneous bacterial infections.

Although extremely effective, the wide use of Chx has increased the awareness of a potential decrease in sensitivity among bacterial isolates, especially in human medicine. , , Because of such risk and the lack of new antibiotics, increased attention has been focused on ways to increase the efficacy of commonly used antimicrobials. This goal has been achieved combining anti‐inflammatory medications to antibiotics, using plant extracts to stimulate the local immune response, , or testing host defence peptides (HDPs) with commonly used antimicrobials. , In particular, Chx has shown a synergistic effect with the HDP human β‐defensin (BD)3 against oral bacteria.

Host defence peptides are an important component of the local innate immunity against microbes. In dogs, several BDs have been identified in epithelial tissues including skin, lungs and urogenital tract. , , , , Among the HDPs studied in dogs, BD103 and cathelicidin (Cath) have the highest antimicrobial activity against a wide range of bacteria. , , Alterations in HDP production and/or secretion have been imputed as the potential reasons why some dogs and people may be more affected by bacterial infections. This theory has been somewhat reinforced by a study showing how skin washes from atopic dogs have a reduced antimicrobial activity compared with skin washes collected from healthy dogs. Furthermore, another study showed how HDPs are more adherent to the surface of atopic skin compared with healthy skin, potentially reducing the concentration of readily available HDPs.

Although Chx is used worldwide as a topical antimicrobial, its antimicrobial interaction with cutaneous innate immune defences is unknown. To answer this question, this study was designed to assess the effects of Chx, at minimal inhibitory concentration (MICs) and sub‐MIC, with and without selected HDPs, against clinical isolates of MDR‐SP.

Methods and materials

Staphylococcal isolates

Ten clinical isolates of MDR‐SP were tested (see Table S1 in Supporting information). The isolates were identified and collected by the clinical microbiology laboratory at the authors’ (DS, KL) institution. Staphylococcal isolates were obtained from skin cultures and processed for routine bacterial culture and sensitivity as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Isolates were identified based on biochemical reactions, using the Trek Sensititre (TREK Diagnostic Systems Inc.; Cleveland, OH, USA) automated system and confirmed via matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectroscopic analysis. Likewise, the MRSP status of the isolates was confirmed via agglutination test for the penicillin bind protein 2a (PBP2a) and confirmed via mecA gene analysis. The MIC was measured by the same system using CLSI guidelines. Although a specific definition of MDR is not present for S. pseudintermedius, isolates were defined as MDR if resistant to at least three classes of antibiotic, based on published guidelines for S. aureus (SA). The American Type Culture Collection (ATCC) S. pseudintermedius (ATCC# 49444) strain was tested as an experimental internal control.

Peptide preparation

For this study two HDPs (cBD103 and cCath) were synthesised (Peptide Protein Research Ltd; Fareham, UK) and tested. Each peptide was prepared as described previously. , , , One milligram of each lyophilised peptide was diluted in 10 mM 0.01%acetic acid to generate a stock concentration of 4 mg/mL; the acetic acid activates the bonds between the cysteine residues without adverse effects on fungi or other bacteria. , , , Each peptide was further diluted (1:2,000) with 10 mM sodium phosphate buffer (SPB) at pH 7.4.

Minimal inhibitory concentration (MIC) assay

For each assay, clinical isolates, and the internal control (ATCC# 49444), were subcultured on nutrient media agar (Himedia; Maharashtra‐Mumbai, India) at 37°C for 24 h. Colonies then were collected and suspended in sterile Nutrient broth (Himedia) to achieve an optical density equal to 0.5 McFarland Standard [˜1 x 108 colony forming units (cfu)/mL] using a Sensititre nephelometer (ThermoFisher Scientific; Waltham, MA, USA). The bacterial suspension then was diluted in sterile SPB (1:100) to obtain a concentration of ˜1 x 106 cfu/mL. All assays were performed in duplicate using sterile 96 well polystyrene round‐bottomed plates (Costar, Corning Inc.; Corning, NY, USA) using the broth microdilution method adapted from CLSI, as reported previously. The experimental error was accepted if falling within the doubling dilution.

Four two‐fold serial dilutions [1:32,000 (0.624 µg/mL), 1:64,000 (0.312 µg/mL), 1:128,000 (0.156 µg/mL), and 1:256,000 (0.078 µg/mL)] of a 2% Chx solution (Sigma‐Aldrich; St Louis, MO, USA) were made. Each HDP (cBD103 and cCath) was tested alone at a concentration of 1 µg/mL. Each concentration of Chx also was tested alone or in combination with 1 µg/mL synthetic HDP (cBD103 or cCath) in SPB. The concentrations of Chx to be tested were based on MIC data derived from preliminary data (data not shown) and previous studies on SA and SP. , , , Likewise, the concentration of 1 µg/mL synthetic HDP was based on previous studies on the amount of HDPs secreted in skin washes from healthy and atopic dogs, and the MIC/MBC of both HDPs tested. , , , Each well contained 50 µL antimicrobial (Chx or HDP), 50 µL inoculum (bacteria in SPB), and 50 µL SPB. To test the interactions between Chx and each HDP, 50 µL inoculum was added to 50 µL HDP and 50 µL Chx. As described previously, the inoculum with or without the antimicrobial(s) was incubated for 2 h in the absence of broth. Once all the samples were collected for the time–kill experiments (see MBC assays section below), 50 µL Nutrient broth (Himedia) was added to each well. Negative control wells contained 50 µL Nutrient broth (Himedia) without bacteria and 100 µL SPB, whereas positive control wells contained 50 µL inoculum and 100 µL SPB. The plates were incubated at 35°C for 18–20 h and the MIC recorded. The MIC was defined as the lowest concentration of antimicrobial with no visible pellets.

Minimal bactericidal concentration (MBC) assays

After 5, 15, 30 and 60 min incubation, 1 µL bacterial suspension (Speed Streaks, Hardy Diagnostics; Santa Maria, CA, USA) at each tested dilution and controls, after energetic stirring, was plated in duplicate,on nutrient media agar and incubated at 35°C for 24 h. The MBC was defined as the lowest dilution at which micro‐organisms were no longer viable on subculture.

Time‐kill method

The median log10 values of the individual colony counts of bacteria recovered from each antimicrobial concentration at each time point (5, 15, 30 and 60 min) were collected and represented graphically. The time–kill method was used and adapted to calculate the interaction between Chx and HDPs. Using this method, synergy was defined as a 2‐log10 decrease in colony count at each time point by the combination compared with the colony count of the most active single agent (Chx). Additivity or indifference was defined as 1‐log10 decrease in colony count at each time point by the combination compared with the colony count of the most active single agent (Chx). Antagonism was defined as a 2‐log10 increase in colony count at each time point by the combination compared with the colony count of the most active agent alone (Chx).

Statistical analysis

Statistical analysis was applied to the MBC data only, comparing the combination of Chx with HDPs and Chx alone at the same concentration of Chx and the same time points. The collected data first were tested for normal distribution using the Shapiro–Wilks test (α = 0.05). Then, Friedman’s test was performed to evaluate the behaviour of each data variable in each group at each time point. If statistically significant, a Dunn’s Multiple Comparison Test was performed as post hoc analysis. A P‐value of ≤0.05 was considered statistically significant. All statistical comparisons were performed using prism v6 (GraphPad Software Inc.; La Jolla, CA, USA).

Results

MIC assays

After 24 h of incubation, Chx alone showed an MIC of 0.312 µg/mL in nine of 10 isolates (MIC90) with a single isolate (ID: 276) having an MIC of <0.078 µg/mL. Likewise, an MIC90 of 0.312 µg/mL was achieved when the bacteria were incubated with the combination of Chx and cBD103. More precisely, MICs were achieved of 0.312 µg/mL in six isolates, 0.156 µg/mL in two isolates, and 0.624 µg/mL in one isolate (ID: 42400) and <0.078 µg/mL in another (ID: 276). When bacteria were incubated with Chx and cCath in combination, an MIC90 was achieved at a concentration of 0.312 µg/mL Chx; notwithstanding this, three isolates had an MIC of 0.156 µg/mL and one (ID: 276) had an MIC of <0.078 µg/mL. However, the wells containing the HDPs alone showed growth for each isolate without reaching an MIC. The negative controls showed no bacterial growth, while the positive controls showed growth for each isolate. The ATCC strain had an MIC of <0.078 µg/mL for the Chx alone or in combination with either HDP, although an MIC was not reached with the HDPs alone.

MBC assays

After 30 and 60 min of incubation with Chx alone, an MBC of 0.624 µg/mL was found in only one isolate (ID: 41781). An MBC was not achieved for any other clinical isolates and any time point. The wells containing the HDPs alone showed growth for most of the clinical isolates tested without reaching an MBC; one isolate (ID: 41781) reached an MBC after 5 min of incubation with cBD103, while a second isolate (ID: 349) reached an MBC at 60 min of incubation. As far as cCath, only one isolate (ID: 46046) reached an MBC after 60 min of incubation. The MBCs for the combinations of Chx and cBD103, and of Chx and cCath varied among isolates, different concentrations of Chx, and time points.

When the colonies recovered from the combination of Chx and cBD103 were compared to the colonies recovered from Chx alone, a significant decrease in colony count was observed at 15 min (0.624 µg/mL; P = 0.04) and 60 min (0.312 µg/mL; P = 0.03). However, when the colonies recovered from the combination of Chx and cCath were compared to the colonies recovered from Chx alone, a significant decrease in colony count was observed in multiple concentrations at multiple time points except after 15 min of incubation (0.15625 µg/mL; P = 0.09) (Figure 1). Like the MIC, the negative controls showed no bacterial growth, while the positive controls showed growth for each isolate. After 60 min of incubation with Chx alone, the ATCC strain had an MBC of 0.321 µg/mL. However, an MBC of <0.078 µg/mL was achieved for bacteria incubated for 15 min in the combination of Chx and cBD103, and for 60 min in the combination of Chx and cCath.

Figure 1

Median of the colony forming units (cfu) recovered after 5, 15, 30 and 60 min incubation with chlorhexidine digluconate (Chx) and host defence peptides (HDPs) alone or in combination.

Groups were compared using Friedman’s test with Dunn's multiple comparison test. *: comparison with Chx 0.625 μg/mL (*, P ≤ 0.05; **,P ≤ 0.01); ^: comparison with Chx 0.312 μg/mL (^, P ≤ 0.05; ^^, ≤ 0.01;   ≤ 0.001); #: comparison with Chx 0.156 μg/mL (#, P ≤ 0.05; ##, P ≤ 0.01); v: comparison with Chx 0.0.078 μg/mL (v, P ≤ 0.05; vv, P ≤ 0.01). HDPs: cBD103, canine β‐defensin 103; cCath, canine cathelicidin. Lines on bars indicate upper and lower quartiles.

Time–kill method

A reduction of ≥1‐log10 was seen for the combination of Chx and cCath at most of the concentrations and time points tested (Figure 2). A 2‐log10 reduction was achieved for the combination of Chx and cCath used at the highest concentrations of Chx after 30 min (0.625 µg/mL) and 60 min (0.625 µg/mL and 0.312 µg/mL) of incubation. A lack of effect was observed with the combination of Chx and cBD103.

Figure 2

Time–kill curves of chlorhexidine digluconate (Chx) and host defence peptide (HDP) alone or in combination.

**, synergy; ^, additivity/neutrality. HDPs: cBD103, canine β‐defensin 103; cCath, canine cathelicidin. Positive indicates bacteria incubated in absence of antimicrobials (positive control).

Discussion

This is the first study in veterinary medicine demonstrating a synergistic/additive effect between Chx and HDPs. The MoA of such combinations is not clear. However, one potential MoA could involve the membrane‐disruptive action of one antimicrobial (e.g. HDPs) on Staphylococci making the other (e.g. Chx) more effective. In fact, both HDPs tested here and Chx are cationic antimicrobials whose major MoAs are the binding to and disruption of bacterial membranes. , ,

The exact amounts of readily available (released) HDPs on the cutaneous surface currently are unknown rendering it difficult to decide the correct amount of HDP to be tested. The choice of using a concentration of 1 µg/mL HDP was based on previous in vitro and in vivo studies. , , , , , Such studies showed an amount of ≤0.4 µg/mL HDPs secreted in the skin wash of healthy and atopic dogs. However, because of the increased adhesion of HDPs to the stratum corneum demonstrated in canine skin, the amount of HDPs is likely to be greater than the one secreted in skin washes. Finally, the MIC/MBC of cBD103 and cCath for methicillin‐resistant SP has been found to be ˜25 µg/mL, a much greater concentration than those recovered in skin washes. Thus, based on these studies, a sub‐MIC/MBC concentration of 1 µg/mL HDPs was selected as this was reasonably present on canine skin. However, it is noteworthy to mention that in vivo a multitude of antimicrobial molecules work together against multiple pathogens.

Likewise, the range of concentrations tested for Chx was based on previous studies on SA and SP. , , , In particular, a starting working concentration of 0.625 µg/mL was chosen based on the high variability on the antimicrobial action of Chx shown against SA (0.625–250 µg/mL) and SP (7 µg/mL). , , , To assess a potential synergy between Chx and HDPs, several sublethal dilutions of Chx were selected. It was found that higher concentrations of Chx resulted in a bactericidal effect of Chx, not allowing further comparison between Chx alone and the combinations of Chx and HDP.

In order to assess the potential synergy between Chx and HDPs, a time–kill method was selected. This method was chosen because it is very flexible and better suited to the assessment of MBCs over time. Because of the short contact time achieved by Chx formulations in practice, an incubation time of ≤60 min was selected, yet a potentially extended time–kill curve, encompassing 10 days of exposure, would have accounted for the residual effect of Chx demonstrated in some studies. , , Based on this method, the results of this study show that a synergic effect is present between Chx and cCath, and not CHx and cBD103. However, an additive/neutral effect was seen between Chx and both HDPs tested for most of the concentrations and times analysed. These results are in line with a previous study showing an enhanced antimicrobial effect of human BD3 (human orthologue of cBD103) when associated with Chx. This seemingly positive association between Chx and HDPs, within the narrow concentration range as used in this manuscript, should now be assessed for a wider range of bacterial lineages.

One limitation of this study is the lack of epidemiological characterisation of the bacterial isolates. However, although a multilocus sequence typing (MLST) characterisation was not performed, based on the current knowledge on MRSP epidemiology, it is likely that all of the isolates belonged to the same type.

In conclusion, these preliminary data show the potentiation of Chx’s antimicrobial effects against MDR‐SP when associated with cCath and cBD103. The degree of the potentiation depends on the concentration of Chx, the incubation time and the HDP tested. How this phenomenon works in nature is hard to determine, yet it is possible to speculate that Chx activity when in contact with the skin and the naturally secreted HDPs may increase its efficacy and kill speed. This would explain the positive clinical benefit of Chx against MDR bacterial pyoderma in dogs. Beyond the purpose of this study is the potential direct effect of Chx on HDPs. It would be interesting to test whether or not Chx (with or without an antifungal agent) increases the presence and antimicrobial effect of natural canine HDPs, or if the combination of Chx and HDPs would increase the susceptibility of Chx‐resistant organisms.

Author contributions

Domenico Santoro: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing‐original draft, Writing‐review & editing. Lopamudra Kher: Data curation, Investigation, Writing‐review & editing. Vanessa Chala: Conceptualization, Writing‐review & editing. Christelle Navarro: Conceptualization, Writing‐review & editing.

Table S1. Susceptibility of Staphylococcus pseudintermedius clinical isolates, n = 10. MDR is defined by methicillin resistance and resistance to at least one agent (bold) in three or more antimicrobial categories. ,

Click here for additional data file.