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Susceptibility of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to chlorhexidine digluconate, octenidine dihydrochloride, polyhexanide, PVP-iodine and triclosan in comparison to hospital-acquired MRSA (HA-MRSA) and community-aquired MRSA (CA-MRSA): a standardized comparison

Susceptibility of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to... Background: Recent publications have raised concerns of reduced susceptibilities of clinical bacterial isolates towards biocides. This study presents a comparative investigation of the susceptibility of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA), hospital-acquired MRSA (HA-MRSA) and community-aquired MRSA (CA-MRSA) to the commonly used antiseptics chlorhexidine (CHX), octenidine (OCT), polyhexanide (PHMB), PVP-iodine (PVP-I) and triclosan (TCX) based on internationally accepted standards. Methods: In total, 28 (18 LA-, 5 HA- and 5 CA) genetically characterized MRSA strains representing a broad spectrum of hosts, clonal complexes and spa-types, as well as the reference methicillin-sensitive Staphylococcus aureus (MSSA) strain ATCC 6538, were selected. Minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MBC) were determined in accordance with DIN 58940–7, 58940–8 and DIN EN ISO 20776-1. The microbicidal efficacy was determined in accordance with DIN EN 1040. Results: Results from the MIC/MBC and quantitative suspension tests revealed differences between antiseptic substances but not between epidemiological groups of MRSA strains. OCT and PHMB were the most active substances with a minimal MIC of 1 mg/L, followed by CHX (2 mg/L), TCX (32 mg/L) and finally PVP-I (1024 mg/L). The MSSA reference strain showed a tendency to a higher susceptibility compared to the MRSA strains. (Continued on next page) * Correspondence: Kathleen.dittmann@uni-greifswald.de Institute of Hygiene and Environmental Medicine, University Medicine of Greifswald, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany University Medicine of Greifswald, Institute of Hygiene and Environmental Medicine, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 2 of 9 (Continued from previous page) Conclusions: This investigation of the susceptibility of a range of LA-, HA- and CA-MRSA strains using standardized conditions gave no indication that LA-MRSA strains are less susceptible to commonly used antiseptics compared to HA- and CA-MRSA strains. Keywords: MRSA, Resistance, Decolonization, Antisepsis Background The S. aureus Genotyping Kit 2.0 (Alere Technologies Antiseptic agents such as chlorhexidine digluconate (chlor- GmbH, Jena, Germany) was used to test selected isolates hexidine, CHX), octenidine dihydrochloride (OCT), poly- for the presence of genes encoding quaternary ammo- hexanide (polyhexamethylene biguanide, PHMB), PVP- nium compound efflux pumps (qac genes), as described iodine (Poly(vinylpyrrolidone)-iodine complex, PVP-I), and elsewhere [25]. None of the tested strains in this com- triclosan (5-chlorine-2-(2,4-dichlorphenoxy)-phenol, TCX) parison harbored qac genes. are widely used as topical antiseptics against colonization and infection of humans and animals with Methicillin- Test preparations resistant Staphylococcus aureus (MRSA) [1–7]. The clinical Chlorhexidine digluconate (20% CHX solution, C 9394, benefits of decolonization of MRSA patients for prevention Sigma-Aldrich Biochemie GmbH, Hamburg, Germany), of nosocomial infections is well documented [8–10]. The octenidine dihydrochloride (Schülke & Mayr GmbH, antimicrobial properties of these agents against hospital Norderstedt, Germany), polyhexanide (20% PHMB acquired (HA) MRSA strains have been repeatedly shown solution, Fagron GmbH & Co. KG, Hamburg, Germany) [11–17]. However, to our best knowledge, there are no and PVP-I (Betaisodona solution: 100 ml of the solution systematic investigations comparing the susceptibility of contains 10 g of poly(1-vinyl-2-pyrrolidone-)iodine- livestock-associated (LA) and community-associated (CA) complex, with a content of 11% available iodine, Mundi- strains versus HA-MRSA strains to these antiseptics using pharma GmbH, Limburg, Germany) and were diluted in standardized and harmonized test procedures. As CA- and water of standardized hardness (WSH; according to DIN LA-MRSA strains make up a growing proportion of MRSA EN 1040 [19]) to the final test concentrations. As TCX strainsinhumans[18], such studies are quite pertinent. (Irgasan, 72779, Fluka, Buchs, Switzerland) dissolves Our investigation was to test different antiseptics to se- poorly in water, a stock solution of 50% TCX in 80% lected MRSA strains reflecting stains that are prevalent in dimethylsulfoxide (DMSO) was diluted in several steps Germany with the main attention on LA-MRSA. to obtain a final concentration of 1% TCX in 40% DMSO/WSH. A 40% DMSO/WSH solution was used in Methods all dilution steps. In order to provide reliable and reproducible informa- The suitability of 40% DMSO in WSH was demon- tion on the susceptibility of MRSA strains, the minimal strated using the quantitative suspension test and the inhibitory concentration (MIC), the minimal microbici- microdilution test as described previously [26]. dal concentration (MBC) (microdilution test; EN 58940) The following solutions were used as neutralizing agents as well as the microbicidal efficacy (quantitative suspen- in accordance with DIN EN 1040 and 1275 [19, 20]: sion test; EN 1040) of CHX, OCT, PHMB, PVP-I and TCX were determined in a comparative study under – 3.0% (w/v) polysorbate 80 + 3.0% (w/v) saponin + standardized conditions [19–22] using a spectrum of 0.1% (w/v) L-histidine + 0.1% (w/v) cysteine for neu- genetically characterized strains from different hosts. tralizing CHX, OCT and PHMB – 3.0% (w/v) polysorbate 80 + 0.3% (w/v) lecithin + Test strains 0.3% (w/v) L-histidine + 0.5% (w/v) sodium thiosul- Strains were drawn from the national collection of the fate for neutralizing PVP-I Robert Koch-Institute (RKI) to represent a broad – 8.0% (w/v) polysorbate 80 + 2.0% (w/v) sodium spectrum of strains from different hosts that are preva- dodecylsulfate (SDS) + 0.8% (w/v) lecithin + 1.0% lent in Germany, and from a collection of regional (w/v) sodium thiosulfate + 6.0% (w/v) saponin for strains from northeastern Germany (HICARE Study) neutralizing TCX. (Table 1)[23]. Methicillin-sensitive Staphylococcus aur- eus (MSSA) ATCC 6538 was used as the reference To determine the MICs and the MBCs, the substances strain. The reference strain comes from the American were prepared in concentrations from 0.25 to 4096 mg/L Type Culture Collection (ATCC®), a scientifically recog- (Table 2). Concentration ranges used in the quantitative nized source, and has defined resistance properties [24]. suspension tests are summarized in Table 2. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 3 of 9 Table 1 List of LA-MRSA, HA-MRSA and CA-MRSA strains with source, spa-type, SCCmec, resistance phenotype and provider LA-MRSA Source Spa-type SCCmec; other Resistance Phenotype Provider CC398 pig t034 V PEN, OXA, ERY, CLI, TET, CIPi, SXT, OXA/Su RKI CC398 cow t011 ND PEN, OXA, TET, OXA/Su RKI CC398 turkey t034 ND PEN, OXA, ERY, CLI, TET, SXTi, OXA/Su RKI CC398 poultry t011 ND PEN, OXA, ERY, CLI, TET, SXT, OXA/Su RKI CC398 horse t011 IV PEN, OXA, GEN, ERY, CLI, TET, CMP, SXT, OXA/Su RKI CC398 horse t6867 IV PEN, OXA, GEN, TET, COX, OXA/Su RKI CC398 human t034 V PEN, OXA, TET, SXTi, OXA/Su RKI CC398 human t899 IV PEN, OXA, OXA/Su RKI CC398 human t2123 ND PEN, OXA, GEN, TET, CIP, OXA/Su RKI CC398 human t2370 ND ND HICARE CC398 human t1456 ND ND HICARE CC398 human t3275 ND ND HICARE CC398 human t10721 ND ND HICARE CC130 deer t843 ND PEN, OXA, OXA/Su RKI CC130 horse t843 ND PEN, OXA, OXA/Su RKI CC130 human t1773 ND PEN, OXA, CIPi, OXA/Su RKI CC9 chicken t1430 ND PEN, OXA, CIP, MFL, OXA/Su RKI CC9 human t1430 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/SU RKI HA-MRSA Source Spa-type SCCmec; other Provider CC22 human t032 IV PEN, OXA, ERY, CLI, CMP, CIP, MFL, OXA/Su RKI CC22 human t020 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/Su RKI CC22 human t005 ND PEN, OXA, OXA/Su RKI CC5 (ST225) human t003 II PEN, OXA, ERY, CLI, CMP, CIP, MFL, OXA/Su RKI CC5 (ST5) human t002 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/Su RKI CA-MRSA Source Spa-type SCCmec; other Provider CC1 human t5100 nd, lukPV, seh PEN, OXA, GEN, TET, FUS, COX, OXA/Su RKI CC8 human t1476 ND ND HICARE CC8 human t008 IV, lukPV PEN, OXA, ERY, CIP, MFL, OXA/Su RKI CC80 human t044 IV, lukPV PEN, OXA, TET, CIP, MUPi, FUS, OXA/Su RKI CC59 human t437 nd, lukPV PEN, OXA, ERY, CLI, TET, CMP, OXA/Su RKI RKI Robert Koch-Institute, HICARE HICARE Study, ND not determinded; Groups of strains were defined genetically by spa-typing, MLST, and SCCmec, as well as demonstration of luk-PV Microdilution test DIN EN 58940–7[21] and 58940–8[22] and the corre- sponding supplementary sheets were strictly followed to Table 2 Concentration ranges used for determining MICs and determine the MIC and MBC, as described previously MBCs in accordance with DIN EN 58940–7 and 58940–8) [18] [26]. Briefly, the test organisms were cultivated on and concentration ranges of the test preparations used in the CASO agar at 37 °C for 18 h; thereafter, four to five col- quantitative suspension tests according to DIN EN 1040 [15] onies were transferred into 1 ml of BBL Mueller Hinton CHX OCT PHMB PVP-I TCX Broth (BD, Becton Dickinson) and diluted to reach 5 × Concentration range of Antiseptic agent for MICs and MBCs 10 cfu/ml. Tests were performed in 96-well microtiter determination [mg/L] plates. Each test was performed in duplicate. Each well 0.25–4 0.25–4 0.25–4 256–4096 16–256 was filled with 100 μl of defined antiseptic dilution and Concentration range of Antiseptic agents for quantitative suspension 100 μl of test organism suspension. The turbidity was tests [mg/L] visually evaluated as an indicator of bacterial growth and 125–500 20–40 50–100 5,000 – 10,000 250–1,000 minimal inhibitory concentration after 24 h (MIC ) and 24 Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 4 of 9 after 48 h (MIC ). To determine the MBC, samples in Statistics the range of the threshold for turbidity after 24 h were Data were prepared using Microsoft Excel 2010 (Micro- transferred onto blood agar, as described in the stan- soft, Redmond, WA, USA) and analyzed using IBM dards, and evaluated for growth after 24 h incubation SPSS Statistics 24 (IBM, Armonk, NY, USA). Strains (MBK ). were grouped as LA-, HA- and CA strains. Robust nonparametric statistics were used to compare results from the microdilution tests and quantitative Quantitative suspension test suspension tests [27]. A two-step procedure was chosen DIN EN 1040 [19] was strictly applied to determine the to avoid alpha-error inflation. Kruskal-Wallis tests were bactericidal efficacy without organic load. Briefly, 0.1 ml used as omnibus tests for multiple comparisons. If the of test organism suspension and 0.1 ml of WSH were omnibus tests indicated statistically significant differ- mixed and left for 2 min. Afterwards, 0.8 ml of the re- ences between groups, Mann-Whitney tests were used spective antiseptic test substance were added. The for pairwise comparisons. resulting solutions were incubated for 5 and 30 min at 37 °C. At the end of the contact time, 0.1 ml of the test Results solution was transferred to 0.8 ml of the respective neu- MIC and MBC tralizing solution and 0.1 ml WSH and left for 5 min. Values of MICs and MBCs of tested substances showed Serial dilutions were prepared in neutralizer; 0.1 ml of marked differences between LA-, HA- and CA-MRSA each neutralized test dilution was spread onto nutrient (Table 3). OCT and PHMB were the most active sub- agar plates in duplicates. After incubation for 24 h, the stances with a minimum MIC of 1 mg/L followed by colonies were counted and the number of recoverable CHX, TCX and finally PVP-I. There was no significant colonies (N ) in the test solution was calculated. The re- a difference between MIC and MIC of the same 24 48 duction factor (RF) was determined as the difference of substances between LA-, HA- and CA-MRSA (Related- the log number of cells in the test solution at the begin- Samples-Wilcoxon-Signed-Rank Test, p = 1.00). There ning of the contact time (N ) and log of N . 0 a was a significant differences between MBC and MIC 24 24 In addition to the DIN EN, negative controls using for all substances but TCX (Related-Samples-Wilcoxon- 0.8 ml of WSH instead of test preparation were per- Signed-Rank-Test, p < 0.01). TCX showed the greatest formed simultaneously in the first test run to exclude range between minimum and maximum MIC and MBC any bactericidal effects of WSH. In the water con- values. trols, no essential difference was observed compared Values of MIC ,MIC and MBC differed signifi- 24 48 24 to the N values. cantly between groups of strains for PHBM (p = 0.003, Table 3 Rounded means and range of MIC , MIC and MBC LA-, HA- and CA-MRSA strains in mg/L 24 48 Stains ATCC_6538 LA-MRSA HA-MRSA CA-MRSA Total (reference) Substance and Test Mean Mean Range Mean Range Mean Range Mean Range p-Value CHX MIC 42 2–43 2–43 2–43 2–4 0.217 CHX MIC 42 2–43 2–43 2–43 2–4 0.217 CHX MBC 85 4–84 4–45 4–85 4–8 0.173 OCT MIC 21 1–21 1–11 1–21 1–2 0.153 OCT MIC 21 1–21 1–11 1–21 1–2 0.153 OCT MBC 42 2–42 2–23 2–43 2–4 0.153 PHMB MIC 12 1–22 2–22 2–22 1–2 0.003 PHMB MIC 12 1–22 2–22 2–22 1–2 0.003 PHMB MBC 14 2–44 4–44 4–44 1–4 0.002 PVP-I MIC 1024 1991 1024–4096 2048 2048–2048 2458 2048–4096 2048 1024–4096 0.098 PVP-I MIC 1024 1991 1024–4096 2048 2048–2048 2458 2048–4096 2048 1024–4096 0.098 PVP-I MBC 2048 3754 2048–4096 4096 4096–4096 4096 4096–4096 3814 2048–4096 0.053 TCX MIC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 TCX MIC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 TCX MBC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 24 Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 5 of 9 p = 0.003 and p = 0.002) and TCX (all p = 0.02) but not susceptibility of the targeted pathogens to the antisep- for CHX (p = 0.217, p = 0.217 and p = 0.173), OCT (all tic products used. p = 0.153) and PVP-I (p = 0.098, p = 0.098 and p = 0.053) Other mechanisms for reduced susceptibility to disin- in the Independent-Samples Kruskal-Wallis test. Pair- fectants in MRSA besides the qac gene coded efflux wise comparison of MIC , MIC and MBC of PHMB pumps have been described: reduced susceptibility to 24 48 24 showed that this was caused by a higher susceptibility of chlorhexidine can also result from mutations in the the reference MSSA strain compared to the MRSA norA/norB genes which code for an efflux mechanism strains (p < 0.02). Pairwise comparisons for TCX showed [29]. Reduced susceptibility to triclosan can be due to that HA- and CA-MRSA strains were less susceptible to either enhanced expression of the target of this biocide, TCX than the reference strain (p = 0.021 and 0.01 re- namely the enoyl-acyl carrier protein (ACP) reductase spectively), and CA-MRSA was significantly less suscep- enzyme (FabI) [37], or acquisition of an additional sh- tible than LA-MRSA (p = 0.035). fabI allele derived from Staphylococcus haemolyticus by horizental gene transfer [27]. We found no evidence of reduced susceptibility of LA-MRSA to CHX, OCT, Quantitative suspension test PHMB, PVP-I and TCX in comparison to CA- and HA- The archived reduction factors show that all substances MRSA. Differences in the susceptibility between the were used at or below the concentration needed to strains in MIC, MBC and microdilution assays were achieve the threshold set by DIN EN 1040 (at least a 5 marginal. With a difference not greater than one dilution log-step reduction) to be adequately bactericidal, as step, the range between the highest and lowest MIC and planned. As expected, reduction factors increased with MBC between the groups of MRSA stains was at the contact time and concentration of the antiseptic same level or even smaller, as between the strains of the (Fig. 1a-e). In contrast, the MSSA reference strain same group (one step for CHX, PHMB and OCT and up showed a higher susceptibility to CHX than did the to two steps for PVP-I and TCX). The only exception MRSA strains, but the differences were not statistically was TCX in terms of LA-MRSA strains, which were significant in the omnibus test. All other tests for statis- significantly more susceptible than CA-MRSA. tical significance were omitted due to the small absolute Likewise, the results from the quantitative suspension differences and the overlapping confidence intervals. assays were quite comparable between CA-, HA- and CA-MRSA strains. In contrast, the reference MSSA Discussion strain showed a tendency to higher susceptibility in the The antibacterial activity of common antiseptics against MIC, MBC and quantitative suspension assays. However, a broad range of different pathogens has been well docu- as only one reference strain was used, it is unclear mented [26, 28]. Still, little is known about the differ- whether this can be interpreted as higher susceptibility ences in the susceptibility to antiseptics of LA-MRSA in of MSSA in contrast to MRSA or as an attribute of the comparison to HA-MRSA and CA-MRSA. While anti- specific strain. septics show a broader antimicrobial spectrum com- Our results are well comparable with those of other pared to antibiotics and are less compromised by published studies. MICs reported by Koburger et al. for specific resistances, reduced susceptibility of various aureus ATCC 6538 almost matched our results, with the strains to antiseptics has been reported [29–33]. Besides exception of PVP-I and TCX, which showed a markedly antimicrobial agents other facts like metal-resistance higher MIC and MBC in our tests [26]. The differ- 48 24 genes might contribute to differences the susceptibility ences for PVP-I remain unexplained, while the reported to antiseptics. For example Argudin et al. reported the higher MICs to TCX in comparison to Koburger et al. occurrence of different metal-resistance genes among (0.125 versus 8 mg/L) can be explained by the fact that LA-MRSA [31]. Recent publications in particular have 8 mg/L was the lowest concentration used in our tests. raised concerns of reduced susceptibilities of distinctive Furthermore, the tested MRSA-strain, a northern clinical isolates towards biocides and found associations German epidemic strain, showed susceptibilities com- with outbreaks [29, 33, 34]. parable to our results. Likewise, MICs to PHMB and Therefore, the susceptibility of LA-MRSA to antisep- TXC reported by Assadian et al. for MRSA, low level tics is an important issue, as LA-MRSA is an emerging vancomycin-resistant (VISA) S. aureus strains and S. problem and antiseptic agents are valuable drugs for aureus ATCC 29213 correspond well to our results [11]. prevention of MRSA infections [18, 35]. For example, Interestingly, the MSSA reference strains showed a ten- antiseptic decolonization has been proven to control the dency to higher susceptibility to TCX in this two studies spread of MRSA in intensive care healthcare settings [8] compared to MRSA. and to reduce surgical site infections [36]. Neverthe- It is important to bear in mind that the concentrations less, the effectiveness of these measures relies on the used in our study were well below the concentrations Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 6 of 9 Fig. 1 (See legend on next page.) Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 7 of 9 (See figure on previous page.) Fig. 1 a Results of quantitative suspension test for chlorhexidine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of chlorhexidine (CHX; 125 mg/L, 250 mg/L and 500 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. b Results of quantitative suspension test for octinidine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of octinidine (OCT; 50 mg/L, 75 mg/L and 100 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. c Results of quantitative suspension test for polyhexanide for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of polyhexanide (PHMB; 5000 mg/L, 7500 mg/L and 10000 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. d Results of quantitative suspension test for PVP-iodine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of PVP-iodine (PVP-I; 20 mg/L, 30 mg/L and 40 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. e Results of quantitative suspension test for triclosan for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of triclosan (TCX; 250 mg/L, 500 mg/L and 1000 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040 recommended by the manufacturer. For example, PHMB evaluate the susceptibility of LA-MRSA to different anti- is used at a concentration of 0.02% or 200 mg/L, which septics in comparison to HA-MRSA and CA-MRSA. is 200 times greater than the MIC for wound The MSSA reference strain serves as an intern control. antisepsis. The shown difference between the reference and the test The strength of the present study is the systematic ap- strains should not be interpreted as evidence for a higher proach based both on European standards for assessing susceptibility of MSSA to MRSA strains in general. the bactericidal effects in quantitative suspension assays Regarding the limited number of antiseptics used, we and on industry standards to determine the MIC and covered a broad spectrum of substances with different MBC using the microdilution method [19, 21, 22, 26]. modes of action. Our selection included CHX, probably the Our method can therefore easily be replicated by other most commonly used antiseptic agent worldwide, and researchers and for other strains. One point worthy of OCT, PHMB, PVP-I and TCX. These substances are widely note is that parts of DIN 58940 have since been sus- used in specific fields of application, such as antisepsis on pended and replaced by DIN EN ISO 20776-1:2007–02. skin and mucous membranes [1, 4], the eye [42, 43], acute However, this has no effects in terms of determining the and chronic wounds [2, 6]and sutures[44]. MIC and MBC for antiseptics in this study. In summary, the present study gives no reason to Our study has limitations. For instances, we used only doubt that the tested antiseptics can kill LA-MRSA at a limited number of strains and antiseptics for our ana- the concentrations recommended for use by the manu- lysis. It is well known that some strains express higher facturer. However, if the substances are diluted, which resistances to specific antiseptics. Resistance to antisep- can happen deliberately as result of the usage (e.g., when tics can arise through different mechanisms [38]. For irrigating wounds) or as part of the intended application example, efflux-mediated resistance to various biocides (e.g., slow release of CHX from patches or TCX from linked to qac-genes has been reported in different sutures), the concentration may be reduced to levels that staphylococcal isolates in recent years [39–41]. However, fall short of the MIC. As recent publications raise con- this does not detract from our research question of cerns about the increasing resistance of clinical isolates whether LA-strains show a higher resistance to antisep- to antiseptics and disinfectants, this highlights the im- tics compared to HA- and CA strains per se, as qac- portance of safe and conscientious use of antiseptics. genes have been reported in HA-, CA- and LA-strains alike. Although we used a limited number of strains, all Conclusion were genetically characterized and represented a broad This investigation of the susceptibility of a broad range spectrum of hosts, clonal complexes and spa-types. Most of HA-, LA- and CA-MRSA strains using standardized strains were drawn from the national collection of the and harmonized conditions provided no indication Robert Koch Institute and were supplemented by re- that LA-MRSA strains show reduced susceptibility to gional strains from northeastern Germany as well as an commonly used antiseptics compared to HA- and ATCC reference strain. The aim of our study was to CA-MRSA strains. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 8 of 9 Abbrevations 5. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine CA-MRSA: Community-aquired Methicillin-resistant Staphylococcus aureus; in endodontics. Int Endod J. 2009;42(4):288–302. CHX: Chlorhexidine; HA-MRSA: Hospital-acquired Methicillin-resistant 6. Sopata M, Ciupinska M, Glowacka A, Muszynski Z, Tomaszewska E. Effect of Staphylococcus aureus; LA-MRSA: Livestock-associated Methicillin-resistant Octenisept antiseptic on bioburden of neoplastic ulcers in patients with Staphylococcus aureus; MBC: Minimal microbicidal concentration; advanced cancer. J Wound Care. 2008;17(1):24–7. MIC: Minimal inhibitory concentration; MRSA: Methicillin-resistant 7. Novakov Mikic A, Budakov D. Comparison of local metronidazole and a Staphylococcus aureus; MSSA: Methicillin-sensitive Staphylococcus aureus; local antiseptic in the treatment of bacterial vaginosis. Arch Gynecol Obstet. OCT: Octenidine; PHMB: Polyhexanide; PVP-I: (Poly(vinylpyrrolidone)-iodine 2010;282(1):43–7. complex); TCX: Triclosan 8. Huang SS, Septimus E, Kleinman K, Moody J, Hickok J, Avery TR, et al. Targeted versus universal decolonization to prevent ICU infection. N Engl J Acknowledgements Med. 2013;368(24):2255–65. Not applicable. 9. Musuuza JS, Guru PK, O'Horo JC, Bongiorno CM, Korobkin MA, Gangnon RE, et al. The impact of chlorhexidine bathing on hospital-acquired Authors’ contributions bloodstream infections: a systematic review and meta-analysis. BMC Infect NOH and KD conceived the study and its original design. DT, TS and CC Dis. 2019;19(1):416. collected the data. TS, DT, GM and SH were responsible for the 10. Gebreselassie HM, Lo Priore E, Marschall J. Effectiveness of meticillin-resistant microbiological testing. KD, NH and GM drafted the manuscript supported Staphylococcus aureus decolonization in long-term haemodialysis patients: a by all authors. All authors read and approved the final manuscript. systematic review and meta-analysis. J Hosp Infect. 2015;91(3):250–6. 11. Assadian O, Wehse K, Hubner NO, Koburger T, Bagel S, Jethon F, et al. Funding Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) This research was conducted with the financial support of BBraun AG of polihexanide and triclosan against antibiotic sensitive and resistant (Melsungen, Germany). The sponsors did not participate in any way in Staphylococcus aureus and Escherichia coli strains. GMS Krankenhaushygiene conducting the study or interpreting the results. interdisziplinar. 2011;6(1):Doc06. 12. Muller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Availability of data and materials Chemother. 2008;61(6):1281–7. The data and materials are available from the corresponding author on 13. Koburger T, Muller G, Eisenbeiss W, Assadian O, Kramer A. Microbicidal reasonable request. activity of polihexanide. GMS Krankenhaushyg Interdiszip. 2007;2(2):Doc44. 14. Al-Doori Z, Morrison D, Goroncy-Bermes P, Edwards G, Gemmell C. Susceptibility Ethics approval and consent to participate of MRSA to Octenidine Dihydrochloride. ECCMID Congress; Nice 2006. For used human samples of the HICARE Study ethical approval from the 15. Pitten FA, Werner HP, Kramer A. A standardized test to assess the impact of ethics committee of University Medicine of Greifswald (BB 07/12, BB 111/12) different organic challenges on the antimicrobial activity of antiseptics. J was sought. For the other human samples with the clonal complex CC398 Hosp Infect. 2003;55(2):108–15. ethical approval from the ethics committee of the medical faculty of 16. Sakuragi T, Yanagisawa K, Dan K. Bactericidal activity of skin disinfectants on University Magdeburg (# 47/09) was sought. For the remaining human methicillin-resistant Staphylococcus aureus. Anesth Analg. 1995;81(3):555–8. samples ethical approval was not nessesary because this samples were 17. Haley CE, Marling-Cason M, Smith JW, Luby JP, Mackowiak PA. Bactericidal transmittal of clinic-diagnostic laboratories. activity of antiseptics against methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 1985;21(6):991–2. Consent for publication Not applicable. 18. van Alen S, Ballhausen B, Peters G, Friedrich AW, Mellmann A, Kock R, et al. In the Centre of an epidemic: fifteen years of LA-MRSA CC398 at the university hospital Munster. Vet Microbiol. 2017;200:19–24. Competing interests 19. DIN EN 1040 Chemical disinfectants and antiseptics - Quantitative Peter Pfaff is an employee of BBraun AG GmbH (Melsungen, Germany). The suspension test for the evaluation of basic bactericidal activity of chemical antiseptic compounds CHX and PHMB are part of some of the products of disinfectants and antiseptics - Test method and requirements (phase 1) BBraun AG GmbH. The other authors declare that they have no competing German version EN 1040:2005. Sect. 03 (2006). interests. None of the authors holds stock or options in BBraun AG GmbH. 20. DIN EN 1275 Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic fungicidal or basic yeasticidal Author details activity of chemical disinfectants and antiseptics - Test method and Institute of Hygiene and Environmental Medicine, University Medicine of requirements (phase 1); German version EN 1275:2005. Sect. 03 (2006). Greifswald, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany. Robert 21. DIN 58940–7 Medical microbiology - Susceptibility testing of microbial Koch Institute, Unit 13: Nosocomial Pathogens and Antibiotic Resistances, pathogens to antimicrobial agents - Part 7: Determination of the minimum Burgstraße 37, 38855 Wernigerode, Germany. Department of Immunology, bactericidal concentration (MBC) with the method of microbouillondilution; University of Greifswald, Ferdinand-Sauerbruch-Str, 17475 Greifswald, Text in German and English. Sect. 08 (2009). Germany. BBraun AG, Carl-Braun-Straße 1, 34212 Melsungen, Germany. University Medicine of Greifswald, Institute of Hygiene and Environmental 22. DIN 58940–8 Medical microbiology - Susceptibility testing of pathogens to Medicine, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany. antimicrobial agents - Part 8: Microdilution; General method-specific requirements. Sect. 10 (2002). Received: 6 March 2019 Accepted: 17 July 2019 23. Gerlich MG, Piegsa J, Schafer C, Hubner NO, Wilke F, Reuter S, et al. Improving hospital hygiene to reduce the impact of multidrug-resistant organisms in health care--a prospective controlled multicenter study. BMC Infect Dis. 2015;15:441. References 24. ATCC: The Global Bioresource Center 2019 [Available from: https://www. 1. Durani P, Leaper D. Povidone-iodine: use in hand disinfection, skin atcc.org/]. preparation and antiseptic irrigation. Int Wound J. 2008;5(3):376–87. 25. Monecke S, Jatzwauk L, Muller E, Nitschke H, Pfohl K, Slickers P, et al. 2. Daeschlein G, Assadian O, Bruck JC, Meinl C, Kramer A, Koch S. Feasibility Diversity of SCCmec elements in Staphylococcus aureus as observed in and clinical applicability of polihexanide for treatment of second-degree south-eastern Germany. PLoS One. 2016;11(9):e0162654. burn wounds. Skin Pharmacol Physiol. 2007;20(6):292–6. 3. Kampf G, Kramer A. Epidemiologic background of hand hygiene and 26. Koburger T, Hubner NO, Braun M, Siebert J, Kramer A. Standardized evaluation of the most important agents for scrubs and rubs. Clin Microbiol comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine Rev. 2004;17(4):863–93 table of contents. dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob 4. Dogan AA, Cetin ES, Hussein E, Adiloglu AK. Microbiological evaluation of Chemother. 2010;65(8):1712–9. octenidine dihydrochloride mouth rinse after 5 days' use in orthodontic 27. Ciusa ML, Furi L, Knight D, Decorosi F, Fondi M, Raggi C, et al. A novel patients. Angle Orthod. 2009;79(4):766–72. resistance mechanism to triclosan that suggests horizontal gene transfer Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 9 of 9 and demonstrates a potential selective pressure for reduced biocide susceptibility in clinical strains of Staphylococcus aureus. Int J Antimicrob Agents. 2012;40(3):210–20. 28. Kramer A, Dissemond J, Kim S, Willy C, Mayer D, Papke R, et al. Consensus on wound antisepsis: update 2018. Skin Pharmacol Physiol. 2018;31(1):28–58. 29. Hardy K, Sunnucks K, Gil H, Shabir S, Trampari E, Hawkey P, et al. Increased usage of antiseptics is associated with reduced susceptibility in clinical isolates of Staphylococcus aureus. MBio. 2018;9(3):e00894–18. 30. Lee H, Lim H, Bae IK, Yong D, Jeong SH, Lee K, et al. Coexistence of mupirocin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates from Korea. Diagn Microbiol Infect Dis. 2013;75(3):308–12. 31. Kampf G, Jarosch R, Ruden H. Limited effectiveness of chlorhexidine based hand disinfectants against methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect. 1998;38(4):297–303. 32. Wand ME, Bock LJ, Bonney LC, Sutton JM. Mechanisms of Increased Resistance to Chlorhexidine and Cross-Resistance to Colistin following Exposure of Klebsiella pneumoniae Clinical Isolates to Chlorhexidine. Antimicrob Agents Chemother. 2017;61(1):e01162–16. 33. Pidot SJ, Gao W, Buultjens AH, Monk IR, Guerillot R, Carter GP, et al. Increasing tolerance of hospital Enterococcus faecium to handwash alcohols. Sci Transl Med. 2018;10(452). 34. Wassilew N, Seth-Smith HM, Rolli E, Fietze Y, Casanova C, Fuhrer U, et al. Outbreak of vancomycin-resistant Enterococcus faecium clone ST796, Switzerland, December 2017 to April 2018. Euro Surveill. 2018;23(29):1800351. 35. Goerge T, Lorenz MB, van Alen S, Hubner NO, Becker K, Kock R. MRSA colonization and infection among persons with occupational livestock exposure in Europe: prevalence, preventive options and evidence. Vet Microbiol. 2017;200:6–12. 36. Sadigursky D, Pires HS, Rios SAC, Rodrigues Filho FLB, Queiroz GC, Azi ML. Prophylaxis with nasal decolonization in patients submitted to total knee and hip arthroplasty: systematic review and meta-analysis. Rev Bras Ortop. 2017;52(6):631–7. 37. Grandgirard D, Furi L, Ciusa ML, Baldassarri L, Knight DR, Morrissey I, et al. Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression. BMC Genomics. 2015;16:345. 38. Russell AD. Mechanisms of bacterial insusceptibility to biocides. Am J Infect Control. 2001;29(4):259–61. 39. Worthing KA, Marcus A, Abraham S, Trott DJ, Norris JM. Qac genes and biocide tolerance in clinical veterinary methicillin-resistant and methicillin- susceptible Staphylococcus aureus and Staphylococcus pseudintermedius. Vet Microbiol. 2018;216:153–8. 40. Smith K, Gemmell CG, Hunter IS. The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates. J Antimicrob Chemother. 2008;61(1):78–84. 41. Seier-Petersen MA, Nielsen LN, Ingmer H, Aarestrup FM, Agerso Y. Biocide susceptibility of Staphylococcus aureus CC398 and CC30 isolates from pigs and identification of the biocide resistance genes, qacG and qacC. Microb Drug Resist. 2015;21(5):527–36. 42. Hübner NO, Kramer A. Review on the efficacy, safety and clinical applications of Polihexanide, a modern wound antiseptic. Skin Pharmacol Physiol. 2010;23:17–27. 43. Ristau T, Kirchhof B, Fauser S. Antisepsis with polyhexanide is effective against endophthalmitis after intravitreal injections. Acta Ophthalmol. 2014; 92(6):e494–6. 44. Sandini M, Mattavelli I, Nespoli L, Uggeri F, Gianotti L. Systematic review and meta-analysis of sutures coated with triclosan for the prevention of surgical site infection after elective colorectal surgery according to the PRISMA statement. Medicine. 2016;95(35):e4057. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Resistance & Infection Control Springer Journals

Susceptibility of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to chlorhexidine digluconate, octenidine dihydrochloride, polyhexanide, PVP-iodine and triclosan in comparison to hospital-acquired MRSA (HA-MRSA) and community-aquired MRSA (CA-MRSA): a standardized comparison

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Biomedicine; Medical Microbiology; Drug Resistance; Infectious Diseases
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10.1186/s13756-019-0580-9
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Abstract

Background: Recent publications have raised concerns of reduced susceptibilities of clinical bacterial isolates towards biocides. This study presents a comparative investigation of the susceptibility of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA), hospital-acquired MRSA (HA-MRSA) and community-aquired MRSA (CA-MRSA) to the commonly used antiseptics chlorhexidine (CHX), octenidine (OCT), polyhexanide (PHMB), PVP-iodine (PVP-I) and triclosan (TCX) based on internationally accepted standards. Methods: In total, 28 (18 LA-, 5 HA- and 5 CA) genetically characterized MRSA strains representing a broad spectrum of hosts, clonal complexes and spa-types, as well as the reference methicillin-sensitive Staphylococcus aureus (MSSA) strain ATCC 6538, were selected. Minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MBC) were determined in accordance with DIN 58940–7, 58940–8 and DIN EN ISO 20776-1. The microbicidal efficacy was determined in accordance with DIN EN 1040. Results: Results from the MIC/MBC and quantitative suspension tests revealed differences between antiseptic substances but not between epidemiological groups of MRSA strains. OCT and PHMB were the most active substances with a minimal MIC of 1 mg/L, followed by CHX (2 mg/L), TCX (32 mg/L) and finally PVP-I (1024 mg/L). The MSSA reference strain showed a tendency to a higher susceptibility compared to the MRSA strains. (Continued on next page) * Correspondence: Kathleen.dittmann@uni-greifswald.de Institute of Hygiene and Environmental Medicine, University Medicine of Greifswald, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany University Medicine of Greifswald, Institute of Hygiene and Environmental Medicine, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 2 of 9 (Continued from previous page) Conclusions: This investigation of the susceptibility of a range of LA-, HA- and CA-MRSA strains using standardized conditions gave no indication that LA-MRSA strains are less susceptible to commonly used antiseptics compared to HA- and CA-MRSA strains. Keywords: MRSA, Resistance, Decolonization, Antisepsis Background The S. aureus Genotyping Kit 2.0 (Alere Technologies Antiseptic agents such as chlorhexidine digluconate (chlor- GmbH, Jena, Germany) was used to test selected isolates hexidine, CHX), octenidine dihydrochloride (OCT), poly- for the presence of genes encoding quaternary ammo- hexanide (polyhexamethylene biguanide, PHMB), PVP- nium compound efflux pumps (qac genes), as described iodine (Poly(vinylpyrrolidone)-iodine complex, PVP-I), and elsewhere [25]. None of the tested strains in this com- triclosan (5-chlorine-2-(2,4-dichlorphenoxy)-phenol, TCX) parison harbored qac genes. are widely used as topical antiseptics against colonization and infection of humans and animals with Methicillin- Test preparations resistant Staphylococcus aureus (MRSA) [1–7]. The clinical Chlorhexidine digluconate (20% CHX solution, C 9394, benefits of decolonization of MRSA patients for prevention Sigma-Aldrich Biochemie GmbH, Hamburg, Germany), of nosocomial infections is well documented [8–10]. The octenidine dihydrochloride (Schülke & Mayr GmbH, antimicrobial properties of these agents against hospital Norderstedt, Germany), polyhexanide (20% PHMB acquired (HA) MRSA strains have been repeatedly shown solution, Fagron GmbH & Co. KG, Hamburg, Germany) [11–17]. However, to our best knowledge, there are no and PVP-I (Betaisodona solution: 100 ml of the solution systematic investigations comparing the susceptibility of contains 10 g of poly(1-vinyl-2-pyrrolidone-)iodine- livestock-associated (LA) and community-associated (CA) complex, with a content of 11% available iodine, Mundi- strains versus HA-MRSA strains to these antiseptics using pharma GmbH, Limburg, Germany) and were diluted in standardized and harmonized test procedures. As CA- and water of standardized hardness (WSH; according to DIN LA-MRSA strains make up a growing proportion of MRSA EN 1040 [19]) to the final test concentrations. As TCX strainsinhumans[18], such studies are quite pertinent. (Irgasan, 72779, Fluka, Buchs, Switzerland) dissolves Our investigation was to test different antiseptics to se- poorly in water, a stock solution of 50% TCX in 80% lected MRSA strains reflecting stains that are prevalent in dimethylsulfoxide (DMSO) was diluted in several steps Germany with the main attention on LA-MRSA. to obtain a final concentration of 1% TCX in 40% DMSO/WSH. A 40% DMSO/WSH solution was used in Methods all dilution steps. In order to provide reliable and reproducible informa- The suitability of 40% DMSO in WSH was demon- tion on the susceptibility of MRSA strains, the minimal strated using the quantitative suspension test and the inhibitory concentration (MIC), the minimal microbici- microdilution test as described previously [26]. dal concentration (MBC) (microdilution test; EN 58940) The following solutions were used as neutralizing agents as well as the microbicidal efficacy (quantitative suspen- in accordance with DIN EN 1040 and 1275 [19, 20]: sion test; EN 1040) of CHX, OCT, PHMB, PVP-I and TCX were determined in a comparative study under – 3.0% (w/v) polysorbate 80 + 3.0% (w/v) saponin + standardized conditions [19–22] using a spectrum of 0.1% (w/v) L-histidine + 0.1% (w/v) cysteine for neu- genetically characterized strains from different hosts. tralizing CHX, OCT and PHMB – 3.0% (w/v) polysorbate 80 + 0.3% (w/v) lecithin + Test strains 0.3% (w/v) L-histidine + 0.5% (w/v) sodium thiosul- Strains were drawn from the national collection of the fate for neutralizing PVP-I Robert Koch-Institute (RKI) to represent a broad – 8.0% (w/v) polysorbate 80 + 2.0% (w/v) sodium spectrum of strains from different hosts that are preva- dodecylsulfate (SDS) + 0.8% (w/v) lecithin + 1.0% lent in Germany, and from a collection of regional (w/v) sodium thiosulfate + 6.0% (w/v) saponin for strains from northeastern Germany (HICARE Study) neutralizing TCX. (Table 1)[23]. Methicillin-sensitive Staphylococcus aur- eus (MSSA) ATCC 6538 was used as the reference To determine the MICs and the MBCs, the substances strain. The reference strain comes from the American were prepared in concentrations from 0.25 to 4096 mg/L Type Culture Collection (ATCC®), a scientifically recog- (Table 2). Concentration ranges used in the quantitative nized source, and has defined resistance properties [24]. suspension tests are summarized in Table 2. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 3 of 9 Table 1 List of LA-MRSA, HA-MRSA and CA-MRSA strains with source, spa-type, SCCmec, resistance phenotype and provider LA-MRSA Source Spa-type SCCmec; other Resistance Phenotype Provider CC398 pig t034 V PEN, OXA, ERY, CLI, TET, CIPi, SXT, OXA/Su RKI CC398 cow t011 ND PEN, OXA, TET, OXA/Su RKI CC398 turkey t034 ND PEN, OXA, ERY, CLI, TET, SXTi, OXA/Su RKI CC398 poultry t011 ND PEN, OXA, ERY, CLI, TET, SXT, OXA/Su RKI CC398 horse t011 IV PEN, OXA, GEN, ERY, CLI, TET, CMP, SXT, OXA/Su RKI CC398 horse t6867 IV PEN, OXA, GEN, TET, COX, OXA/Su RKI CC398 human t034 V PEN, OXA, TET, SXTi, OXA/Su RKI CC398 human t899 IV PEN, OXA, OXA/Su RKI CC398 human t2123 ND PEN, OXA, GEN, TET, CIP, OXA/Su RKI CC398 human t2370 ND ND HICARE CC398 human t1456 ND ND HICARE CC398 human t3275 ND ND HICARE CC398 human t10721 ND ND HICARE CC130 deer t843 ND PEN, OXA, OXA/Su RKI CC130 horse t843 ND PEN, OXA, OXA/Su RKI CC130 human t1773 ND PEN, OXA, CIPi, OXA/Su RKI CC9 chicken t1430 ND PEN, OXA, CIP, MFL, OXA/Su RKI CC9 human t1430 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/SU RKI HA-MRSA Source Spa-type SCCmec; other Provider CC22 human t032 IV PEN, OXA, ERY, CLI, CMP, CIP, MFL, OXA/Su RKI CC22 human t020 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/Su RKI CC22 human t005 ND PEN, OXA, OXA/Su RKI CC5 (ST225) human t003 II PEN, OXA, ERY, CLI, CMP, CIP, MFL, OXA/Su RKI CC5 (ST5) human t002 ND PEN, OXA, ERY, CLI, CIP, MFL, OXA/Su RKI CA-MRSA Source Spa-type SCCmec; other Provider CC1 human t5100 nd, lukPV, seh PEN, OXA, GEN, TET, FUS, COX, OXA/Su RKI CC8 human t1476 ND ND HICARE CC8 human t008 IV, lukPV PEN, OXA, ERY, CIP, MFL, OXA/Su RKI CC80 human t044 IV, lukPV PEN, OXA, TET, CIP, MUPi, FUS, OXA/Su RKI CC59 human t437 nd, lukPV PEN, OXA, ERY, CLI, TET, CMP, OXA/Su RKI RKI Robert Koch-Institute, HICARE HICARE Study, ND not determinded; Groups of strains were defined genetically by spa-typing, MLST, and SCCmec, as well as demonstration of luk-PV Microdilution test DIN EN 58940–7[21] and 58940–8[22] and the corre- sponding supplementary sheets were strictly followed to Table 2 Concentration ranges used for determining MICs and determine the MIC and MBC, as described previously MBCs in accordance with DIN EN 58940–7 and 58940–8) [18] [26]. Briefly, the test organisms were cultivated on and concentration ranges of the test preparations used in the CASO agar at 37 °C for 18 h; thereafter, four to five col- quantitative suspension tests according to DIN EN 1040 [15] onies were transferred into 1 ml of BBL Mueller Hinton CHX OCT PHMB PVP-I TCX Broth (BD, Becton Dickinson) and diluted to reach 5 × Concentration range of Antiseptic agent for MICs and MBCs 10 cfu/ml. Tests were performed in 96-well microtiter determination [mg/L] plates. Each test was performed in duplicate. Each well 0.25–4 0.25–4 0.25–4 256–4096 16–256 was filled with 100 μl of defined antiseptic dilution and Concentration range of Antiseptic agents for quantitative suspension 100 μl of test organism suspension. The turbidity was tests [mg/L] visually evaluated as an indicator of bacterial growth and 125–500 20–40 50–100 5,000 – 10,000 250–1,000 minimal inhibitory concentration after 24 h (MIC ) and 24 Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 4 of 9 after 48 h (MIC ). To determine the MBC, samples in Statistics the range of the threshold for turbidity after 24 h were Data were prepared using Microsoft Excel 2010 (Micro- transferred onto blood agar, as described in the stan- soft, Redmond, WA, USA) and analyzed using IBM dards, and evaluated for growth after 24 h incubation SPSS Statistics 24 (IBM, Armonk, NY, USA). Strains (MBK ). were grouped as LA-, HA- and CA strains. Robust nonparametric statistics were used to compare results from the microdilution tests and quantitative Quantitative suspension test suspension tests [27]. A two-step procedure was chosen DIN EN 1040 [19] was strictly applied to determine the to avoid alpha-error inflation. Kruskal-Wallis tests were bactericidal efficacy without organic load. Briefly, 0.1 ml used as omnibus tests for multiple comparisons. If the of test organism suspension and 0.1 ml of WSH were omnibus tests indicated statistically significant differ- mixed and left for 2 min. Afterwards, 0.8 ml of the re- ences between groups, Mann-Whitney tests were used spective antiseptic test substance were added. The for pairwise comparisons. resulting solutions were incubated for 5 and 30 min at 37 °C. At the end of the contact time, 0.1 ml of the test Results solution was transferred to 0.8 ml of the respective neu- MIC and MBC tralizing solution and 0.1 ml WSH and left for 5 min. Values of MICs and MBCs of tested substances showed Serial dilutions were prepared in neutralizer; 0.1 ml of marked differences between LA-, HA- and CA-MRSA each neutralized test dilution was spread onto nutrient (Table 3). OCT and PHMB were the most active sub- agar plates in duplicates. After incubation for 24 h, the stances with a minimum MIC of 1 mg/L followed by colonies were counted and the number of recoverable CHX, TCX and finally PVP-I. There was no significant colonies (N ) in the test solution was calculated. The re- a difference between MIC and MIC of the same 24 48 duction factor (RF) was determined as the difference of substances between LA-, HA- and CA-MRSA (Related- the log number of cells in the test solution at the begin- Samples-Wilcoxon-Signed-Rank Test, p = 1.00). There ning of the contact time (N ) and log of N . 0 a was a significant differences between MBC and MIC 24 24 In addition to the DIN EN, negative controls using for all substances but TCX (Related-Samples-Wilcoxon- 0.8 ml of WSH instead of test preparation were per- Signed-Rank-Test, p < 0.01). TCX showed the greatest formed simultaneously in the first test run to exclude range between minimum and maximum MIC and MBC any bactericidal effects of WSH. In the water con- values. trols, no essential difference was observed compared Values of MIC ,MIC and MBC differed signifi- 24 48 24 to the N values. cantly between groups of strains for PHBM (p = 0.003, Table 3 Rounded means and range of MIC , MIC and MBC LA-, HA- and CA-MRSA strains in mg/L 24 48 Stains ATCC_6538 LA-MRSA HA-MRSA CA-MRSA Total (reference) Substance and Test Mean Mean Range Mean Range Mean Range Mean Range p-Value CHX MIC 42 2–43 2–43 2–43 2–4 0.217 CHX MIC 42 2–43 2–43 2–43 2–4 0.217 CHX MBC 85 4–84 4–45 4–85 4–8 0.173 OCT MIC 21 1–21 1–11 1–21 1–2 0.153 OCT MIC 21 1–21 1–11 1–21 1–2 0.153 OCT MBC 42 2–42 2–23 2–43 2–4 0.153 PHMB MIC 12 1–22 2–22 2–22 1–2 0.003 PHMB MIC 12 1–22 2–22 2–22 1–2 0.003 PHMB MBC 14 2–44 4–44 4–44 1–4 0.002 PVP-I MIC 1024 1991 1024–4096 2048 2048–2048 2458 2048–4096 2048 1024–4096 0.098 PVP-I MIC 1024 1991 1024–4096 2048 2048–2048 2458 2048–4096 2048 1024–4096 0.098 PVP-I MBC 2048 3754 2048–4096 4096 4096–4096 4096 4096–4096 3814 2048–4096 0.053 TCX MIC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 TCX MIC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 TCX MBC 852 32–64 64 64–64 102 64–256 61 8–256 0.020 24 Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 5 of 9 p = 0.003 and p = 0.002) and TCX (all p = 0.02) but not susceptibility of the targeted pathogens to the antisep- for CHX (p = 0.217, p = 0.217 and p = 0.173), OCT (all tic products used. p = 0.153) and PVP-I (p = 0.098, p = 0.098 and p = 0.053) Other mechanisms for reduced susceptibility to disin- in the Independent-Samples Kruskal-Wallis test. Pair- fectants in MRSA besides the qac gene coded efflux wise comparison of MIC , MIC and MBC of PHMB pumps have been described: reduced susceptibility to 24 48 24 showed that this was caused by a higher susceptibility of chlorhexidine can also result from mutations in the the reference MSSA strain compared to the MRSA norA/norB genes which code for an efflux mechanism strains (p < 0.02). Pairwise comparisons for TCX showed [29]. Reduced susceptibility to triclosan can be due to that HA- and CA-MRSA strains were less susceptible to either enhanced expression of the target of this biocide, TCX than the reference strain (p = 0.021 and 0.01 re- namely the enoyl-acyl carrier protein (ACP) reductase spectively), and CA-MRSA was significantly less suscep- enzyme (FabI) [37], or acquisition of an additional sh- tible than LA-MRSA (p = 0.035). fabI allele derived from Staphylococcus haemolyticus by horizental gene transfer [27]. We found no evidence of reduced susceptibility of LA-MRSA to CHX, OCT, Quantitative suspension test PHMB, PVP-I and TCX in comparison to CA- and HA- The archived reduction factors show that all substances MRSA. Differences in the susceptibility between the were used at or below the concentration needed to strains in MIC, MBC and microdilution assays were achieve the threshold set by DIN EN 1040 (at least a 5 marginal. With a difference not greater than one dilution log-step reduction) to be adequately bactericidal, as step, the range between the highest and lowest MIC and planned. As expected, reduction factors increased with MBC between the groups of MRSA stains was at the contact time and concentration of the antiseptic same level or even smaller, as between the strains of the (Fig. 1a-e). In contrast, the MSSA reference strain same group (one step for CHX, PHMB and OCT and up showed a higher susceptibility to CHX than did the to two steps for PVP-I and TCX). The only exception MRSA strains, but the differences were not statistically was TCX in terms of LA-MRSA strains, which were significant in the omnibus test. All other tests for statis- significantly more susceptible than CA-MRSA. tical significance were omitted due to the small absolute Likewise, the results from the quantitative suspension differences and the overlapping confidence intervals. assays were quite comparable between CA-, HA- and CA-MRSA strains. In contrast, the reference MSSA Discussion strain showed a tendency to higher susceptibility in the The antibacterial activity of common antiseptics against MIC, MBC and quantitative suspension assays. However, a broad range of different pathogens has been well docu- as only one reference strain was used, it is unclear mented [26, 28]. Still, little is known about the differ- whether this can be interpreted as higher susceptibility ences in the susceptibility to antiseptics of LA-MRSA in of MSSA in contrast to MRSA or as an attribute of the comparison to HA-MRSA and CA-MRSA. While anti- specific strain. septics show a broader antimicrobial spectrum com- Our results are well comparable with those of other pared to antibiotics and are less compromised by published studies. MICs reported by Koburger et al. for specific resistances, reduced susceptibility of various aureus ATCC 6538 almost matched our results, with the strains to antiseptics has been reported [29–33]. Besides exception of PVP-I and TCX, which showed a markedly antimicrobial agents other facts like metal-resistance higher MIC and MBC in our tests [26]. The differ- 48 24 genes might contribute to differences the susceptibility ences for PVP-I remain unexplained, while the reported to antiseptics. For example Argudin et al. reported the higher MICs to TCX in comparison to Koburger et al. occurrence of different metal-resistance genes among (0.125 versus 8 mg/L) can be explained by the fact that LA-MRSA [31]. Recent publications in particular have 8 mg/L was the lowest concentration used in our tests. raised concerns of reduced susceptibilities of distinctive Furthermore, the tested MRSA-strain, a northern clinical isolates towards biocides and found associations German epidemic strain, showed susceptibilities com- with outbreaks [29, 33, 34]. parable to our results. Likewise, MICs to PHMB and Therefore, the susceptibility of LA-MRSA to antisep- TXC reported by Assadian et al. for MRSA, low level tics is an important issue, as LA-MRSA is an emerging vancomycin-resistant (VISA) S. aureus strains and S. problem and antiseptic agents are valuable drugs for aureus ATCC 29213 correspond well to our results [11]. prevention of MRSA infections [18, 35]. For example, Interestingly, the MSSA reference strains showed a ten- antiseptic decolonization has been proven to control the dency to higher susceptibility to TCX in this two studies spread of MRSA in intensive care healthcare settings [8] compared to MRSA. and to reduce surgical site infections [36]. Neverthe- It is important to bear in mind that the concentrations less, the effectiveness of these measures relies on the used in our study were well below the concentrations Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 6 of 9 Fig. 1 (See legend on next page.) Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 7 of 9 (See figure on previous page.) Fig. 1 a Results of quantitative suspension test for chlorhexidine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of chlorhexidine (CHX; 125 mg/L, 250 mg/L and 500 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. b Results of quantitative suspension test for octinidine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of octinidine (OCT; 50 mg/L, 75 mg/L and 100 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. c Results of quantitative suspension test for polyhexanide for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of polyhexanide (PHMB; 5000 mg/L, 7500 mg/L and 10000 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. d Results of quantitative suspension test for PVP-iodine for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of PVP-iodine (PVP-I; 20 mg/L, 30 mg/L and 40 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040. e Results of quantitative suspension test for triclosan for LA-MRSA, HA-MRSA, CA-MRSA and reference MSSA. Different concentrations of triclosan (TCX; 250 mg/L, 500 mg/L and 1000 mg/L) were suspended to different MRSA strains and the MSSA reference strain at two different contact times (5 min and 30 min). LA-MRSA (beige), HA-MRSA (green), CA-MRSA (blue) and reference MSSA (purple). Error bars show 95% confidence intervals. Horizontal line at the value of the mean reduction factor of 5 indicates bactericidal threshold according to DIN EN 1040 recommended by the manufacturer. For example, PHMB evaluate the susceptibility of LA-MRSA to different anti- is used at a concentration of 0.02% or 200 mg/L, which septics in comparison to HA-MRSA and CA-MRSA. is 200 times greater than the MIC for wound The MSSA reference strain serves as an intern control. antisepsis. The shown difference between the reference and the test The strength of the present study is the systematic ap- strains should not be interpreted as evidence for a higher proach based both on European standards for assessing susceptibility of MSSA to MRSA strains in general. the bactericidal effects in quantitative suspension assays Regarding the limited number of antiseptics used, we and on industry standards to determine the MIC and covered a broad spectrum of substances with different MBC using the microdilution method [19, 21, 22, 26]. modes of action. Our selection included CHX, probably the Our method can therefore easily be replicated by other most commonly used antiseptic agent worldwide, and researchers and for other strains. One point worthy of OCT, PHMB, PVP-I and TCX. These substances are widely note is that parts of DIN 58940 have since been sus- used in specific fields of application, such as antisepsis on pended and replaced by DIN EN ISO 20776-1:2007–02. skin and mucous membranes [1, 4], the eye [42, 43], acute However, this has no effects in terms of determining the and chronic wounds [2, 6]and sutures[44]. MIC and MBC for antiseptics in this study. In summary, the present study gives no reason to Our study has limitations. For instances, we used only doubt that the tested antiseptics can kill LA-MRSA at a limited number of strains and antiseptics for our ana- the concentrations recommended for use by the manu- lysis. It is well known that some strains express higher facturer. However, if the substances are diluted, which resistances to specific antiseptics. Resistance to antisep- can happen deliberately as result of the usage (e.g., when tics can arise through different mechanisms [38]. For irrigating wounds) or as part of the intended application example, efflux-mediated resistance to various biocides (e.g., slow release of CHX from patches or TCX from linked to qac-genes has been reported in different sutures), the concentration may be reduced to levels that staphylococcal isolates in recent years [39–41]. However, fall short of the MIC. As recent publications raise con- this does not detract from our research question of cerns about the increasing resistance of clinical isolates whether LA-strains show a higher resistance to antisep- to antiseptics and disinfectants, this highlights the im- tics compared to HA- and CA strains per se, as qac- portance of safe and conscientious use of antiseptics. genes have been reported in HA-, CA- and LA-strains alike. Although we used a limited number of strains, all Conclusion were genetically characterized and represented a broad This investigation of the susceptibility of a broad range spectrum of hosts, clonal complexes and spa-types. Most of HA-, LA- and CA-MRSA strains using standardized strains were drawn from the national collection of the and harmonized conditions provided no indication Robert Koch Institute and were supplemented by re- that LA-MRSA strains show reduced susceptibility to gional strains from northeastern Germany as well as an commonly used antiseptics compared to HA- and ATCC reference strain. The aim of our study was to CA-MRSA strains. Dittmann et al. Antimicrobial Resistance and Infection Control (2019) 8:122 Page 8 of 9 Abbrevations 5. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine CA-MRSA: Community-aquired Methicillin-resistant Staphylococcus aureus; in endodontics. Int Endod J. 2009;42(4):288–302. CHX: Chlorhexidine; HA-MRSA: Hospital-acquired Methicillin-resistant 6. Sopata M, Ciupinska M, Glowacka A, Muszynski Z, Tomaszewska E. Effect of Staphylococcus aureus; LA-MRSA: Livestock-associated Methicillin-resistant Octenisept antiseptic on bioburden of neoplastic ulcers in patients with Staphylococcus aureus; MBC: Minimal microbicidal concentration; advanced cancer. J Wound Care. 2008;17(1):24–7. MIC: Minimal inhibitory concentration; MRSA: Methicillin-resistant 7. Novakov Mikic A, Budakov D. Comparison of local metronidazole and a Staphylococcus aureus; MSSA: Methicillin-sensitive Staphylococcus aureus; local antiseptic in the treatment of bacterial vaginosis. Arch Gynecol Obstet. OCT: Octenidine; PHMB: Polyhexanide; PVP-I: (Poly(vinylpyrrolidone)-iodine 2010;282(1):43–7. complex); TCX: Triclosan 8. Huang SS, Septimus E, Kleinman K, Moody J, Hickok J, Avery TR, et al. Targeted versus universal decolonization to prevent ICU infection. N Engl J Acknowledgements Med. 2013;368(24):2255–65. Not applicable. 9. Musuuza JS, Guru PK, O'Horo JC, Bongiorno CM, Korobkin MA, Gangnon RE, et al. The impact of chlorhexidine bathing on hospital-acquired Authors’ contributions bloodstream infections: a systematic review and meta-analysis. BMC Infect NOH and KD conceived the study and its original design. DT, TS and CC Dis. 2019;19(1):416. collected the data. TS, DT, GM and SH were responsible for the 10. Gebreselassie HM, Lo Priore E, Marschall J. Effectiveness of meticillin-resistant microbiological testing. KD, NH and GM drafted the manuscript supported Staphylococcus aureus decolonization in long-term haemodialysis patients: a by all authors. All authors read and approved the final manuscript. systematic review and meta-analysis. J Hosp Infect. 2015;91(3):250–6. 11. Assadian O, Wehse K, Hubner NO, Koburger T, Bagel S, Jethon F, et al. Funding Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) This research was conducted with the financial support of BBraun AG of polihexanide and triclosan against antibiotic sensitive and resistant (Melsungen, Germany). The sponsors did not participate in any way in Staphylococcus aureus and Escherichia coli strains. GMS Krankenhaushygiene conducting the study or interpreting the results. interdisziplinar. 2011;6(1):Doc06. 12. Muller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Availability of data and materials Chemother. 2008;61(6):1281–7. The data and materials are available from the corresponding author on 13. Koburger T, Muller G, Eisenbeiss W, Assadian O, Kramer A. Microbicidal reasonable request. activity of polihexanide. GMS Krankenhaushyg Interdiszip. 2007;2(2):Doc44. 14. Al-Doori Z, Morrison D, Goroncy-Bermes P, Edwards G, Gemmell C. 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Bactericidal transmittal of clinic-diagnostic laboratories. activity of antiseptics against methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 1985;21(6):991–2. Consent for publication Not applicable. 18. van Alen S, Ballhausen B, Peters G, Friedrich AW, Mellmann A, Kock R, et al. In the Centre of an epidemic: fifteen years of LA-MRSA CC398 at the university hospital Munster. Vet Microbiol. 2017;200:19–24. Competing interests 19. DIN EN 1040 Chemical disinfectants and antiseptics - Quantitative Peter Pfaff is an employee of BBraun AG GmbH (Melsungen, Germany). The suspension test for the evaluation of basic bactericidal activity of chemical antiseptic compounds CHX and PHMB are part of some of the products of disinfectants and antiseptics - Test method and requirements (phase 1) BBraun AG GmbH. The other authors declare that they have no competing German version EN 1040:2005. Sect. 03 (2006). interests. None of the authors holds stock or options in BBraun AG GmbH. 20. 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Systematic review and meta-analysis of sutures coated with triclosan for the prevention of surgical site infection after elective colorectal surgery according to the PRISMA statement. Medicine. 2016;95(35):e4057. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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