Background
Antiseptic agents such as chlorhexidine digluconate (chlorhexidine, CHX), octenidine dihydrochloride (OCT), polyhexanide (polyhexamethylene biguanide, PHMB), PVP-iodine (Poly(vinylpyrrolidone)-iodine complex, PVP-I), and triclosan (5-chlorine-2-(2,4-dichlorphenoxy)-phenol, TCX) are widely used as topical antiseptics against colonization and infection of humans and animals with Methicillin-resistant
Staphylococcus aureus (MRSA) [
1‐
7]. The clinical benefits of decolonization of MRSA patients for prevention of nosocomial infections is well documented [
8‐
10]. The antimicrobial properties of these agents against hospital acquired (HA) MRSA strains have been repeatedly shown [
11‐
17]. However, to our best knowledge, there are no systematic investigations comparing the susceptibility of livestock-associated (LA) and community-associated (CA) strains versus HA-MRSA strains to these antiseptics using standardized and harmonized test procedures. As CA- and LA-MRSA strains make up a growing proportion of MRSA strains in humans [
18], such studies are quite pertinent. Our investigation was to test different antiseptics to selected MRSA strains reflecting stains that are prevalent in Germany with the main attention on LA-MRSA.
Discussion
The antibacterial activity of common antiseptics against a broad range of different pathogens has been well documented [
26,
28]. Still, little is known about the differences in the susceptibility to antiseptics of LA-MRSA in comparison to HA-MRSA and CA-MRSA. While antiseptics show a broader antimicrobial spectrum compared to antibiotics and are less compromised by specific resistances, reduced susceptibility of various strains to antiseptics has been reported [
29‐
33]. Besides antimicrobial agents other facts like metal-resistance genes might contribute to differences the susceptibility to antiseptics. For example Argudin et al. reported the occurrence of different metal-resistance genes among LA-MRSA [
31]. Recent publications in particular have raised concerns of reduced susceptibilities of distinctive clinical isolates towards biocides and found associations with outbreaks [
29,
33,
34].
Therefore, the susceptibility of LA-MRSA to antiseptics is an important issue, as LA-MRSA is an emerging problem and antiseptic agents are valuable drugs for prevention of MRSA infections [
18,
35]. For example, antiseptic decolonization has been proven to control the spread of MRSA in intensive care healthcare settings [
8] and to reduce surgical site infections [
36]. Nevertheless, the effectiveness of these measures relies on the susceptibility of the targeted pathogens to the antiseptic products used.
Other mechanisms for reduced susceptibility to disinfectants in MRSA besides the qac gene coded efflux pumps have been described: reduced susceptibility to chlorhexidine can also result from mutations in the norA/norB genes which code for an efflux mechanism [
29]. Reduced susceptibility to triclosan can be due to either enhanced expression of the target of this biocide, namely the enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) [
37], or acquisition of an additional sh-fabI allele derived from Staphylococcus haemolyticus by horizental gene transfer [
27]. We found no evidence of reduced susceptibility of LA-MRSA to CHX, OCT, PHMB, PVP-I and TCX in comparison to CA- and HA-MRSA. Differences in the susceptibility between the strains in MIC, MBC and microdilution assays were marginal. With a difference not greater than one dilution step, the range between the highest and lowest MIC and MBC between the groups of MRSA stains was at the same level or even smaller, as between the strains of the same group (one step for CHX, PHMB and OCT and up to two steps for PVP-I and TCX). The only exception was TCX in terms of LA-MRSA strains, which were significantly more susceptible than CA-MRSA.
Likewise, the results from the quantitative suspension assays were quite comparable between CA-, HA- and CA-MRSA strains. In contrast, the reference MSSA strain showed a tendency to higher susceptibility in the MIC, MBC and quantitative suspension assays. However, as only one reference strain was used, it is unclear whether this can be interpreted as higher susceptibility of MSSA in contrast to MRSA or as an attribute of the specific strain.
Our results are well comparable with those of other published studies. MICs reported by Koburger et al. for aureus ATCC 6538 almost matched our results, with the exception of PVP-I and TCX, which showed a markedly higher MIC
48 and MBC
24 in our tests [
26]. The differences for PVP-I remain unexplained, while the reported higher MICs to TCX in comparison to Koburger et al. (0.125 versus 8 mg/L) can be explained by the fact that 8 mg/L was the lowest concentration used in our tests.
Furthermore, the tested MRSA-strain, a northern German epidemic strain, showed susceptibilities comparable to our results. Likewise, MICs to PHMB and TXC reported by Assadian et al. for MRSA, low level vancomycin-resistant (VISA)
S. aureus strains and
S. aureus ATCC 29213 correspond well to our results [
11]. Interestingly, the MSSA reference strains showed a tendency to higher susceptibility to TCX in this two studies compared to MRSA.
It is important to bear in mind that the concentrations used in our study were well below the concentrations recommended by the manufacturer. For example, PHMB is used at a concentration of 0.02% or 200 mg/L, which is 200 times greater than the MIC24 for wound antisepsis.
The strength of the present study is the systematic approach based both on European standards for assessing the bactericidal effects in quantitative suspension assays and on industry standards to determine the MIC and MBC using the microdilution method [
19,
21,
22,
26]. Our method can therefore easily be replicated by other researchers and for other strains. One point worthy of note is that parts of DIN 58940 have since been suspended and replaced by DIN EN ISO 20776-1:2007–02. However, this has no effects in terms of determining the MIC and MBC for antiseptics in this study.
Our study has limitations. For instances, we used only a limited number of strains and antiseptics for our analysis. It is well known that some strains express higher resistances to specific antiseptics. Resistance to antiseptics can arise through different mechanisms [
38]. For example, efflux-mediated resistance to various biocides linked to qac-genes has been reported in different staphylococcal isolates in recent years [
39‐
41]. However, this does not detract from our research question of whether LA-strains show a higher resistance to antiseptics compared to HA- and CA strains per se, as qac-genes have been reported in HA-, CA- and LA-strains alike. Although we used a limited number of strains, all were genetically characterized and represented a broad spectrum of hosts, clonal complexes and spa-types. Most strains were drawn from the national collection of the Robert Koch Institute and were supplemented by regional strains from northeastern Germany as well as an ATCC reference strain. The aim of our study was to evaluate the susceptibility of LA-MRSA to different antiseptics in comparison to HA-MRSA and CA-MRSA. The MSSA reference strain serves as an intern control. The shown difference between the reference and the test strains should not be interpreted as evidence for a higher susceptibility of MSSA to MRSA strains in general.
Regarding the limited number of antiseptics used, we covered a broad spectrum of substances with different modes of action. Our selection included CHX, probably the most commonly used antiseptic agent worldwide, and OCT, PHMB, PVP-I and TCX. These substances are widely used in specific fields of application, such as antisepsis on skin and mucous membranes [
1,
4], the eye [
42,
43], acute and chronic wounds [
2,
6] and sutures [
44].
In summary, the present study gives no reason to doubt that the tested antiseptics can kill LA-MRSA at the concentrations recommended for use by the manufacturer. However, if the substances are diluted, which can happen deliberately as result of the usage (e.g., when irrigating wounds) or as part of the intended application (e.g., slow release of CHX from patches or TCX from sutures), the concentration may be reduced to levels that fall short of the MIC. As recent publications raise concerns about the increasing resistance of clinical isolates to antiseptics and disinfectants, this highlights the importance of safe and conscientious use of antiseptics.
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