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. 2022 Dec 14;17(12):e0278569. doi: 10.1371/journal.pone.0278569

Chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples in a three-armed cluster randomised decolonisation trial

Luisa A Denkel 1,2,*, Tobias S Kramer 1,2, Frank Schwab 1,2, Jennifer Golembus 1,2, Solvy Wolke 1,2, Petra Gastmeier 1,2, Christine Geffers 1,2
Editor: Surbhi Leekha3
PMCID: PMC9749986  PMID: 36516147

Abstract

Background

Routine use of chlorhexidine or octenidine for antiseptic bathing may have unintended consequences. Our analysis aimed to assess the phenotypic susceptibility of bacterial isolates from clinical samples to chlorhexidine and octenidine collected from intensive care units (ICU) that routinely used 2% chlorhexidine-impregnated wash cloths or 0.08% octenidine wash mitts (intervention) or water and soap (control) for daily patient care.

Methods

This study was conducted within the context of a three armed cluster-randomised controlled decolonisation trial (Registration number DRKS00010475, registration date August 18, 2016). Bacterial isolates were collected prior to and at the end of a 12-month-intervention period from patients with ≥ 3 days length of stay at an ICU assigned to one of two intervention groups or the control group. Phenotypic susceptibility to chlorhexidine and octenidine was assessed by an accredited contract research laboratory determining minimal inhibitory concentrations (MIC) as percentage of extraction solutions used. MIC were reported as estimated concentrations in μg/ml derived from the chlorhexidine and octenidine extraction solutions. Statistical analyses including generalized estimating equation models were applied.

Results

In total, 790 ICU-attributable bacterial isolates from clinical samples (e.g. blood, urine, tracheal aspirate) were eligible for all analyses. Pathogens included were Staphylococcus aureus (n = 155), coagulase-negative staphylococci (CoNS, n = 122), Escherichia coli (n = 227), Klebsiella spp. (n = 150) and Pseudomonas aeruginosa (n = 136). For all species, chlorhexidine and octenidine MIC did not increase from baseline to intervention period in the antiseptic bathing groups. For proportions of bacterial isolates with elevated chlorhexidine / octenidine MIC (≥ species-specific chlorhexidine / octenidine MIC50), adjusted incidence rate ratios (aIRR) showed no differences between the intervention groups and the control group (intervention period).

Conclusion

We found no evidence for reduced phenotypic susceptibilities of bacterial isolates from clinical samples to chlorhexidine or octenidine in ICUs 12 months after implementation of routine antiseptic bathing with the respective substances.

Introduction

Chlorhexidine gluconate and octenidine dihydrochloride are two cationic biocides that can bind covalently to the bacterial cell membrane, subsequently cause depolarization and can lead to bacterial cell death if used at high concentrations [1, 2]. Meta-analyses and clinical trials are available that demonstrate the effectiveness of chlorhexidine bathing to reduce healthcare-associated infections (HAI) including bloodstream infections (BSI) in intensive care units (ICUs) [3–9]. Thus, chlorhexidine is most commonly used for antiseptic bathing of intensive care patients. At the same time, only few clinical trials on the effect of antiseptic bathing with octenidine for BSI prevention are available [10–12]. In Germany, antiseptic bathing of ICU patients is no routine infection control practice. If applied, however, octenidine is the most frequently used substance [13].

We conducted a three-armed cluster-randomised controlled trial (cRCT) in 72 adult ICUs to investigate the effect of daily patient bathing with chlorhexidine, octenidine or routine care with water and soap (control) on central-line associated BSI (CLABSI) rates [14]. This trial found no preventive effect of antiseptic bathing neither with chlorhexidine nor octenidine when comparing CLABSI rates of the intervention groups with the control group [14]. The post hoc before-after analysis of our cluster-randomised decolonisation trial, however, suggests that ICUs with CLABSI rates ≥0.8 CLABSI per 1000 central-line (CL) days might benefit from bathing with 2% chlorhexidine-impregnated cloths [15]. At the same time, octenidine showed no preventive effect on ICU-attributable CLABSI [15].

The use of chlorhexidine or octenidine may have unintended consequences. High usage of these antiseptic substances is associated with increases in chlorhexidine and octenidine minimal inhibitory concentrations (MIC) among clinical isolates [16]. Further, long-term use of chlorhexidine is suspected not only to enhance the development of non-susceptibility, but also to increase antibiotic cross-resistance, decolonization failure or potentially disadvantageous alterations of the skin microbiome [17–20].

Our analysis aimed to answer the question whether daily patient bathing with 2% impregnated chlorhexidine cloths or 0.08% octenidine wash mitts might lead to reduced chlorhexidine or octenidine susceptibilities of ICU-attributable bacterial isolates from clinical samples in the CLIP-ID trial.

Methods

Trial design and participants

This investigation was done within a cluster-randomised controlled trial (cRCT) as part of the project Climate and pathogens–impact of decolonisation (CLIP-ID, registration number DRKS00010475, registration date August 18, 2016). Details of the CLIP-ID trial including the study protocol were published elsewhere [14]. Briefly, our cRCT was conducted on 72 ICUs in 68 hospitals in Germany and Austria. Each ICU represented one cluster and was randomly assigned to one of three decolonisation regimes that had to be applied for 12 consecutive months. The random allocation sequence was computer-generated and applied on the cluster (ICU) level. We stratified ICUs by type of ICU (medical, surgical, interdisciplinary wards in hospitals with < 400 beds and interdisciplinary wards in hospitals with ≥ 400 beds) and size of hospital before randomization. In the chlorhexidine group, all ICUs performed daily patient bathing with 2% chlorhexidine-impregnated cloths (Sage 2% Chlorhexidine Gluconate (CHG) by Stryker) below the jaw line and chlorhexidine-free cloths above the jaw line (Stryker). In the octenidine group, ICUs used 0.08% octenidine disposable wash mitts (Octenisan® by Schülke). In the routine care (control) group, all ICUs applied non-antiseptic soap and water (routine care) for daily patient care.

Collection of the ICU-attributable bacterial isolates from clinical samples

All 72 ICUs participating were requested to collect 10 bacterial isolates from clinical samples prior to and 10 bacterial isolates at the end of the intervention period to analyze phenotypic susceptibility to the antiseptic substances (chlorhexidine and octenidine) applied in this trial. Bacterial isolates collected must met the following inclusion criteria: i) specimen were collected for medical or diagnostic purposes only; ii) bacteria isolated from clinical material (e.g. tracheal aspirate, urine, blood, liquor) not colonization sites (no screening isolates from rectal swabs, stool, nasal-pharyngeal swabs); iii) clinical material from patients with length of stay of at least 3 days on the ICU participating and iv) identification of Staphylococcus (S.) aureus, Enterobacterales (e.g. Escherichia (E.) coli, Klebsiella (K.) subspecies (spp.) or Pseudomonas (P.) aeruginosa from all clinical material or coagulase-negative staphylococci (CoNS) from blood samples only. Bacterial isolates (with Amies transport medium) were sent by the diagnostic laboratories of ICUs participating in the trial to the coordinating study center. The study was not blinded. However, collection of bacterial isolates was done by laboratory personnel who were not informed about assignments of ICUs to one of three study groups. Information on isolates (e.g. ICU name, ICU day of sample collection, anonymous sequential patient number, date, material, and species) were provided by the ICU and their diagnostic laboratories in anonymous form. No patient based data were collected or distributed.

Phenotypic susceptibility to chlorhexidine and octenidine

Phenotypic susceptibility of bacterial isolates from clinical samples to the antiseptic substances contained in the chlorhexidine-impregnated cloths and octenidine disposable wash mitts were analysed by determining minimal inhibitory concentrations (MIC). Phenotypic susceptibility testings were assigned as contract work to an accredited laboratory and conducted according to VAH method 7 (A00040) [21].

Extraction solutions of antiseptic substances (chlorhexidine and octenidine) were harvested by wringing out chlorhexidine-impregnated cloths (Stryker / Sage Products) and octenidine wash mitts (Schülke) provided by the manufacturer. Concentrations of these extraction solutions were estimated to be similar to the concentrations of the ready-to-use products reported by the manufacturers: 2% (20,000 μg/ml) for chlorhexidine and 0.08% (800 μg/ml) for octenidine. Dilutions of antiseptic substances were made in water of standardized water hardness (WSH) according to VAH method 5 [21]. Briefly, bacteria were inoculated in soybean casein digest agar (CSA) at 37°C for 18 – 24h. Concentrations of these bacterial solutions were adjusted to 1.5–5.0 x 108 colony forming units (CFU)/ml. Subsequently, 150 μl of the respective antiseptic dilutions and 150 μl doubled concentrated soybean casein solutions (CSL) were mixed. Microtiter plates were inoculated with 3 μl bacteria (1:10-dilution in CSL) yielding in a final bacterial concentration of 1.5–5.0 x 105 colony forming units (CFU)/ml. Bacteria were inoculated at 37°C for 48 h. The lowest concentration of extraction solutions without bacterial growth (detectable turbidity) was interpreted as minimal inhibitory concentration (MIC) reported in percentage of extraction solution. Percentages were converted to estimated (est.) concentrations of extraction solutions in μg / ml. Controls were inoculated with WSH instead of antiseptic dilutions. Determination of bacteriostatic efficacy and suitable neutralizing agents was done according to VAH method 7 [21]. The following reference strains were included to the analysis: Staphylococcus aureus ATCC6538 (DSM 799), Proteus mirabilis ATCC 14153 (DSM 788), Klebsiella pneumoniae ESBL (DSM 16609), Pseudomonas aeruginosa ATCC 15442 and Escherichia coli NCTC 10538.

Statistical analyses

In the descriptive analysis, numbers with percentages, medians (MIC50) with interquartile ranges (IQR) and / or percentiles (MIC90) were calculated. The variables “sample site” and “study group and study period” were dummy-coded. P-values were calculated by Chi-square test or Fisher’s exact test for categorical, and by Mann-Whitney U Test for continuous variables.

We generated a binary variable to represent phenotypic chlorhexidine and octenidine susceptibilities of bacterial isolates from clinical samples. All bacterial isolates were categorized according to their chlorhexidine and octenidine MIC being ≥ or < the species-specific chlorhexidine and octenidine MIC50 (identified in this study). Bacterial isolates were categorized as “yes” (bacterial isolates with chlorhexidine / octenidine susceptibility ≥ species-specific MIC50) or “no” (chlorhexidine /octenidine susceptibility of bacterial isolates < species-specific MIC50).

In the multivariable analysis, we applied generalised estimating equation (GEE) models to identify effects of chlorhexidine / octenidine interventions on the susceptibility of bacterial isolates from clinical samples to these substances. We used one GEE model for all species to investigate the susceptibility to chlorhexidine, and another GEE model to investigate the susceptibility to octenidine. The outcomes “phenotypic susceptibilities to chlorhexidine / octenidine” were represented by proportions of bacterial isolates with chlorhexidine or octenidine MIC being ≥ the species-specific chlorhexidine / octenidine MIC50. Interaction of study groups (chlorhexidine / octenidine / control) and study periods (baseline / intervention) as well as potential cluster effects were considered in these models [22]. Control group (intervention period) served as reference. Further, ICU day of sample collection and clinical site (blood, tracheal aspirate, urine, wound, or other) were determined as possible confounders and considered in all models. The category “other” as clinical site included e.g. intra-operative samples, intra-abdominal swabs and / or tissue. All parameters added one degree of freedom to the model.

P-values less than 0.05 were considered significant. All analyses were performed using SPSS 27 (IBM SPSS statistics, Somer, NY, USA).

Sample size calculation

Initial sample size calculation of this cRCT was done for the primary outcome CLABSI [14]. However, we performed a power calculation for the outcomes “phenotypic susceptibilities to chlorhexidine / octenidine” represented by proportions of bacterial isolates with chlorhexidine or octenidine MIC being ≥ the species-specific chlorhexidine / octenidine MIC50. The latter were identified in this study (Table 2). We assumed that at the end of the intervention period, 35% of isolates were ≥ MIC50 in the control group (routine care) compared to 65% in the intervention group (antiseptic bathing with chlorhexidine or octenidine). Further, we assumed a ratio of 1:1 between the groups. With individual randomization, a sample size of 70 bacterial isolates in each group would have been required to show the difference with a power of 80%, and a two-sided type 1 error of 0.025. Adjusting for cluster effects with 13 clusters per arm and an intracluster correlation coefficient of 0.1 (adapted from literature [23]), we must have included 101 bacterial isolates per arm. Multiple comparisons to the routine care group were considered by a two-sided type 1 error of 0.025.

Table 2. Chlorhexidine and octenidine minimal inhibitory concentrations (MIC) of ICU-attributable bacterial isolates from clinical samples reported by study group and period.
Total Chlorhexidine group Octenidine group Routine care group = control
Baseline Intervention Baseline Intervention Baseline Intervention
Staphylococcus aureus
n (%) 155 (100.0%) 38 (25.0%) 22 (14.1%) 24 (15.4%) 18 (11.5%) 23 (14.7%) 30 (19.2%)
Chlorhexidine
MIC50 in est. [μg/ml] (IQR) 5.0 (2.0–5.0) 5.0 (5.0–5.0) 2.0 (2.0–4.3) 5.0 (2.0–5.0) 2.0 (2.0–5.0) 5.0 (2.0–5.0) 5.0 (2.8–10.0)
MIC90 in est. [μg/ml] 5.0 5.0 4.3 5.0 5.0 5.0 10.0
Octenidine
MIC50 in est. [μg/ml] (IQR) 2.0 (2.0–2.0) 2.0 (2.0–4.0) 2.0 (0.8–2.0) 2.0 (2.0–2.0) 2.0 (2.0–2.0) 2.0 (2.0–4.0) 2.0 (2.0–2.0)
MIC90 in est. [μg/ml] 2.0 4.0 2.0 2.0 2.0 4.0 2.0
Coagulase-negative staphylococci
n (%) 122 (100.0%) 31 (25.4%) 31 (25.4%) 17 (13.9%) 11 (9.0%) 15 (12.3%) 17 (13.9%)
Chlorhexidine
MIC50 in est. [μg/ml] (IQR) 5.0 (2.0–5.0) 5.0 (2.0–5.0) 2.0 (1.0–2.0) 5.0 (5.0–5.0) 2.0 (1.0–3.5) 5.0 (2.0–5.0) 5.0 (5.0–5.0)
MIC90 in est. [μg/ml] 5.0 5.0 2.0 5.0 3.5 5.0 5.0
Octenidine
MIC50 in est. [μg/ml] (IQR) 2.0 (2.0–4.0) 2.0 (2.0–4.0) 2.0 (0.8–2.0) 2.0 (2.0–4.0) 0.8(0.8–2.0) 4.0 (2.0–4.0) 2.0 (2.0–2.0)
MIC90 in est. [μg/ml] 4.0 4.0 2.0 4.0 2.0 4.0 2.0
Escherichia coli
n (%) 227 (100.0%) 64 (28.2%) 37 (16.3%) 41 (18.1%) 18 (7.9%) 39 (17.2%) 28 (11.9%)
Chlorhexidine
MIC50 in est. [μg/ml] (IQR) 5.0 (2.0–5.0) 5.0 (2.0–5.0) 2.0 (2.0–5.0) 5.0 (5.0–5.0) 2.0 (2.0–5.0) 5.0 (5.0–5.0) 5.0 (2.0–10.0)
MIC90 in est. [μg/ml] 5.0 5.0 5.0 5.0 5.0 5.0 10.0
Octenidine
MIC50 in est. [μg/ml] (IQR) 4.0 (2.0–4.0) 4.0 (4.0–4.0) 2.0 (2.0–4.0) 4.0 (4.0–4.0) 2.0 (2.0–4.0) 4.0 (4.0–4.0) 4.0 (2.0–4.0)
MIC90 in est. [μg/ml] 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Klebsiella spp.
n (%) 150 (100.0%) 28 (18.7%) 28 (18.7%) 23 (15.3%) 29 (19.3%) 20 (13.3%) 22 (14.7%)
Chlorhexidine
MIC50 in est. [μg/ml] (IQR) 50.0 (20.0–50.0) 50.0 (20.0–50.0) 20.0 (20.0–50.0) 50.0 (50.0–50.0) 50.0 (20.0–50.0) 50.0 (42.5–50.0) 50.0 (20.0–50.0)
MIC90 in est. [μg/ml] 50.0 50.0 50.0 50.0 50.0 50.0 50.0
Octenidine
MIC50 in est. [μg/ml] (IQR) 4.0 (4.0–8.0) 4.0 (4.0–12.0) 4.0 (4.0–4.0) 8.0 (4.0–10.0) 4.0 (4.0–4.0) 4.0 (4.0–13.0) 4.0 (4.0–4.0)
MIC90 in est. [μg/ml] 8.0 12.0 4.0 10.0 4.0 13.0 4.0
Pseudomonas aeruginosa
n (%) 136 (100.0%) 19 (14.0%) 18 (13.2%) 17 (12.5%) 40 (29.4%) 18 (13.2%) 24 (17.6%)
Chlorhexidine
MIC50 in est. [μg/ml] (IQR) 50.0 (50.0–50.0) 50.0 (35.0–50.0) 50.0 (50.0–50.0) 50.0 (50.0–50.0) 50.0 (50.0–50.0) 50.0 (50.0–50.0) 50.0 (50.0–50.0)
MIC90 in est. [μg/ml] 50.0 50.0 50.0 50.0 50.0 50.0 50.0
Octenidine
MIC50 in est. [μg/ml] (IQR) 12.0 (8.0–24.0) 8.0 (8.0–20.0) 8.0 (8.0–16.0) 16.0 (8.0–16.0) 12.0 (8.0–16.0) 24.0 (8.0–32.0) 16.0 (8.0–24.0)
MIC90 in est. [μg/ml] 24.0 20.0 16.0 16.0 16.0 32.0 24.0
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Median MIC (MIC50) with inter quartile ranges (IQR) and MIC90 are given in estimated concentrations (est. μg/ml) derived from extraction solutions that were harvested from the ready-to-use products. MIC are reported for Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa in total and by study group (chlorhexidine, octenidine, routine care) and period (baseline, prior to the intervention period, intervention, at the end of the intervention period). Percentages of bacterial isolates per study group and period refer to the total number per species.

Ethics approval

The institutional ethical review board (IRB) of the Charité Universitätsmedizin Berlin granted their approval including a waiver of informed consent for this trial (processing number EA1/093/16). The present study did not include any individual patient data, but analysed aggregated and anonymous data only.

Results

Intervention periods lasted from February 1, 2017 to January 31, 2018 in the octenidine and control group, and from June 1, 2017 to May 31, 2018 in the chlorhexidine group. Baseline periods included 12 months before the interventions started in each study group.

In total, 996 bacterial isolates from clinical samples were collected from participating wards. Among them, 797 bacterial isolates were eligible for analyses of susceptibility to chlorhexidine and octenidine (Fig 1).

Fig 1. Flow chart of ICU-attributable bacterial isolates from clinical samples eligible for analyses of susceptibilities to chlorhexidine and octenidine and included to descriptive analyses.

Fig 1

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CoNS, coagulase-negative staphylococci, ICU, intensive care unit. spp., subspecies.

The selected pathogens detected from ICU-attributable clinical isolates were E. coli (n = 227), S. aureus (n = 155), Klebsiella spp. (n = 150), P. aeruginosa (n = 136), CoNS (n = 122), Proteus spp. (n = 5) and Enterobacter spp. (n = 2). Due to the small number of isolates per species, Proteus spp. and Enterobacter spp. were excluded from all further analyses (Fig 1). In consequence, 790 clinical isolates from 60 ICUs were included in the descriptive analyses. Among them, 417 isolates were collected during baseline when all wards used water and soap for daily bathing, and 373 isolates were collected at the end of the intervention period. Clinical isolates from baseline and intervention period were available from 42 of the 60 ICUs (70.0%) responding.

Characteristics of bacterial isolates reported by study group and period are shown in Table 1. No differences in sample site or ICU day of sample collection were detected between study groups neither during baseline nor intervention period (Table 1). The same was true for baseline versus intervention period comparisons in each study group except for the chlorhexidine group. In this group, the samples sites differed between baseline and intervention periods for E. coli and Klebsiella spp. (Table 1).

Table 1. Characteristics of ICU-attributable bacterial isolates from clinical samples included in the analyses (n = 790) in total and reported by study group and period.

Total Chlorhexidine group P-valuea Octenidine group P-valuea Routine care group = control P-valuea
Baseline Intervention Baseline Intervention Baseline Intervention
Staphylococcus aureus
n (%) 155 (100.0) 38 (25.0) 22 (14.2) 24 (15.5) 18 (11.5) 23 (14.7) 30 (19.2)
ICU day of sample collectionb, median (IQR) 6 (4–11) 6 (4–8) 6 (4–9) 0.769 7 (5–20) 6 (4–9) 0.084 6 (4–11) 5 (4–9) 0.842
Sample site
Bloodb, n (%) 27 (17.3) 7 (17.9) 4 (18.2) 0.633 3 (12.5) 2 (11.1) 0.639 3 (13.0) 8 (26.7) 0.193
Tracheal aspirateb, n (%) 87 (55.8) 23 (59.0) 16 (72.2) 0.340 14 (58.3) 11 (61.1) 0.856 10 (43.5) 13 (43.3) 0.992
Urineb, n (%) 4 (2.6) 1 (2.6) 0 (0.0) 0.633 0 (0.0) 1 (5.6) 0.420 0 (0.0) 2 (6.7) 0.316
Woundb, n (%) 18 (11.6) 4 (10.3) 1 (4.5) 0.389 3 (12.5) 1 (5.6) 0.420 5 (21.7) 4 (13.3) 0.328
Othersb, n (%) 19 (12.3) 3 (7.7) 1 (4.5) 0.532 4 (16.7) 3 (16.7) 0.665 5 (26.3) 3 (15.8) 0.213
Coagulase-negative staphylococci
n (%) 122 (100.0) 31 (25.4) 31 (25.4) 17 (13.9) 11 (9.0) 15 (12.2) 17 (13.9)
ICU day of sample collectionb, median (IQR) 11 (6–19) 13 (8–18) 10 (5–21) 0.418 17 (9–19) 10 (3–16) 0.154 11 (6–24) 7 (4–17) 0.363
Sample site
Bloodb, n (%) 122 (88.4) 31 (100.0) 31 (100.0) n.d. 17 (100.0) 11 (100.0) n.d 15 (100.0) 17 (100.0) n.d.
Tracheal aspirateb, n (%) 0 (0.0) 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d.
Urineb, n (%) 0 (0.0) 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d.
Woundb, n (%) 0 (0.0) 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d.
Othersb, n (%) 0 (0.0) 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d. 0 (0.0) 0 (0.0) n.d.
Escherichia coli
n (%) 227 (100.0) 64 (28.2) 37 (16.3%) 41 (18.1) 18 (7.9%) 39 (17.2) 28 (11.9)
ICU day of sample collectionb, median (IQR) 11 (6–19) 10 (5–16) 7 (4–20) 0.584 9 (5–20) 11 (5–15) 0.947 7 (4–14) 12 (4–22) 0.154
Sample site
Bloodb, n (%) 16 (7.1) 2 (3.1) 5 (13.5) 0.096 3 (7.3) 1 (5.6) 1.000 2 (5.1) 3 (10.7) 0.642
Tracheal aspirateb, n (%) 87 (38.3) 30 (46.9) 8 (21.6) 0.012* 16 (39.0) 8 (44.4) 0.457 13 (33.3) 12 (42.9) 0.427
Urineb, n (%) 50 (22.0) 8 (12.5) 13 (35.1) 0.007* 11 (26.8) 4 (22.2) 1.000 10 (25.6) 4 (14.3) 0.259
Woundb, n (%) 33 (14.5) 8 (12.5) 6 (16.2) 0.405 3 (7.3) 4 (22.2) 0.184 8 (20.5) 4 (14.3) 0.512
Othersb, n (%) 41 (18.1) 16 (25.0) 5 (13.5) 0.131 8 (19.5) 1 (5.6) 0.252 6 (15.4) 5 (17.9) 1.000
Klebsiella spp.
n (%) 150 (100.0) 28 (18.7) 28 (18.7) 23 (15.3) 29 (19.3) 20 (13.3) 22 (14.7)
ICU day of sample collectionb, median (IQR) 10 (5–20) 11 (6–26) 8 (5–24) 0.565 12 (6–21) 11 (6–19) 0.712 9 (4–20) 9 (5–16) 0.889
Sample site
Bloodb, n (%) 18 (12.0) 3 (10.7) 5 (17.9) 0.705 1 (4.3) 2 (6.9) 1.000 4 (20.0) 3 (13.6) 0.691
Tracheal aspirateb, n (%) 74 (49.3) 9 (67.0) 15 (53.6) 0.105 12 (52.2) 17 (58.6) 0.642 9 (45.0) 12 (54.5) 0.537
Urineb, n (%) 17 (11.3) 5 (17.9) 5 (17.9) 1.000 2 (8.7) 3 (10.3) 1.000 2 (10.0) 0 (0.0) 0.221
Woundb, n (%) 21 (11.3) 1 (3.6) 2 (7.1) 1.000 5 (21.7) 6 (20.7) 1.000 3 (15.0) 4 (18.2) 1.000
Othersb, n (%) 20 (13.3) 10 (35.7) 1 (3.6) 0.005* 3 (13.0) 1 (3.4) 0.310 2 (10.0) 3 (13.6) 1.000
Pseudomonas aeruginosa
n (%) 136 (100%) 19 (14.0%) 18 (13.2%) 17 (12.5%) 40 (29.4%) 18 (13.2%) 24 (17.6%)
ICU day of sample collectionb, median (IQR) 12 (6–28) 14 (4–33) 15 (9–20) 0.927 6 (5–21) 15 (9–29) 0.319 11 (5–13) 12 (5–25) 0.949
Sample site
Bloodb, n (%) 2 (1.5) 0 (0.0) 0 (0.0) n.d. 0 (0.0) 1 (2.5) 1.000 1 (5.6) 0 (0.0) 0.429
Tracheal aspirateb, n (%) 82 (60.3) 7 (36.8) 12 (66.7) 0.070 12 (70.6) 26 (65.0) 0.682 11 (61.1) 14 (58.3) 0.856
Urineb, n (%) 16 (7.4) 2 (10.5) 2 (11.1) 1.000 2 (11.8) 4 (10.0) 1.000 2 (11.1) 4 (16.7) 0.685
Woundb, n (%) 15 (11.0) 4 (21.1) 2 (11.1) 0.660 1 (5.9) 4 (10.0) 1.000 3 (16.7) 1 (4.2) 0.297
Othersb, n (%) 21 (15.4) 6 (31.6) 2 (11.1) 0.232 22 (11.8) 5 (12.5) 1.000 1 (5–6) 5 (20.8) 0.214
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P-values were calculated by Chi-quare test or Fisher’s exact test for categorical, and by Mann-Whitney U Test for continuous variables.

aP-values were reported for comparisons between baseline and intervention periods for each study group.

bP-Values were not shown for baseline period comparisons and intervention period comparisons of the three study groups because no significant differences were detected between the study groups in the baseline and the intervention period. baseline, prior to the intervention period, intervention, at the end of the intervention period.

P-values < 0.05 were interpreted as significant (*).

Phenotypic susceptibility of bacterial isolates from clinical samples to chlorhexidine and octenidine

MIC50 and MIC90

For all species, phenotypic susceptibility to chlorhexidine depicted as MIC50 and MIC90 in est. μg/ml did not increase comparing baseline and the intervention periods in all study groups, as well as comparing intervention groups with control group at the end of the intervention (Table 2). The same was true for phenotypic susceptibility to octenidine (Table 2).

Phenotypic susceptibility to chlorhexidine and octenidine of commercially available reference strains (S. aureus, E. coli, P. aeruginosa, K. pneumoniae and Proteus mirabilis) are shown in S1 Table. Chlorhexidine and octenidine MIC50 with IQR and MIC90 in percentage of stock solution are given in S2 Table.

Distribution of chlorhexidine and octenidine MIC for all ICU-attributable bacterial isolates from clinical samples

In the intervention groups, the percentages of bacterial isolates with higher chlorhexidine MIC indicating reduced chlorhexidine susceptibility did not increase comparing baseline and intervention periods for all species (Fig 2A–2E). Further, no increase was observed comparing intervention groups with control group (at the end of the intervention period) for all species. The same observations were made for octenidine MIC (Fig 2A–2E).

Fig 2.

Fig 2

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Distribution of chlorhexidine and octenidine MIC for all ICU-attributable bacterial isolates from clinical samples with Staphylococcus aureus (A), coagulase-negative staphylococci (B), Escherichia coli (C), Klebsiella spp. (D) and Pseudomonas aeruginosa (E). MIC were reported as estimated minimal inhibitory concentrations (est. MIC) in [μg/ml] derived from the respective extraction solutions. ICU-attributable bacterial isolates from clinical samples were collected prior to (baseline, light grey bars) and at the end of the intervention period (dark grey bars) applying 2%-chlorhexidine impregnated cloths (chlorhexidine group), 0.08% octenidine wash mitts (octenidine group) or water and soap (routine care = control).

Interestingly, ICUs in the control group that continued bathing with water and soap collected some bacterial isolates with high chlorhexidine and / or octenidine MIC during intervention period even though no antiseptic substances were applied. High chlorhexidine MIC were found in the baseline period and / or in control group for some isolates of S. aureus, E. coli, Klebsiella spp. and P. aeruginosa and high octenidine MIC for P. aeruginosa (Fig 2A and 2C–2E).

Among the 155 S. aureus isolates, 23 (14.8%) had chlorhexidine MIC ≥ 8μg/ml, while this was the case for 10 of 122 CoNS (8.2%). High chlorhexidine MIC (≥ 64 μg/ml) were rare in E. coli and Klebsiella spp. but frequent in P. aeruginosa (≥ 50 μg/ml, Fig 2C–2E).

Characteristics of all 23 S. aureus isolates with chlorhexidine MIC ≥ 8μg/ml can be found in S3 Table.

Multivariable analyses

We used GEE models to investigate the impact of antiseptic bathing with chlorhexidine or octenidine on the chlorhexidine and octenidine susceptibility of bacterial isolates (all species). Characteristics of bacterial isolates (all species) stratified by chlorhexidine or octenidine MIC ≥ species-specific MIC50 (yes / no) are shown in S4 Table. aIRR for the chlorhexidine and the octenidine models are depicted in Table 3. The proportion of bacterial isolates with MIC ≥ species-specific chlorhexidine and octenidine MIC50 did not significantly differ between chlorhexidine or octenidine intervention periods compared with the control group (intervention period, Table 3). We detected significant differences between study groups and study periods with our GEE models (Table 3). However, these differences seemed to be independent from antiseptic bathing. Further, we found evidence that ICU day of sample collection was significantly associated with increased proportions of bacterial isolates with MIC ≥ species-specific MIC50 for chlorhexidine, but not for octenidine. Interestingly, bacterial isolates with chlorhexidine MIC ≥ species-specific chlorhexidine MIC50 were significantly less likely to originate from blood compared with other sample sites. This observation was not found for octenidine.

Table 3. Generalized estimating equation models for the outcomes chlorhexidine and octenidine susceptibility of bacterial isolates (reported by MIC ≥ species-specific chlorhexidine and octenidine MIC50 (yes / no)).

Chlorhexidine
aIRR 95%CI p-value p-value (type III)
Chlorhexidine group in the baseline period 0.85 0.31–2.35 0.771 < 0.001*
Chlorhexidine group in the intervention period 0.29 0.12–0.74 0.010*
Octenidine group in the baseline period 1.81 0.69–4.78 0.229
Octenidine group in the intervention period 0.67 0.25–1.79 0.428
Control group in the baseline period 1.05 0.49–2.23 0.908
Control group in the intervention period 1.00 = reference - -
ICU day of sample collection 1.01 1.00–1.03 0.013 0.013*
Sample site 0.015*
Blood 0.36 0.19–0.70 0.002
Tracheal aspirate 0.73 0.45–1.17 0.187
Urine 0.82 0.43–1.57 0.548
Wound 0.79 0.41–1.51 0.470
Others 1.00 = reference
Octenidine
Chlorhexidine group in the baseline period 1.27 0.56–2.91 0.570 < 0.001*
Chlorhexidine group in the intervention period 0.52 0.25–1.07 0.085
Octenidine group in the baseline period 2.83 1.19–6.76 0.019*
Octenidine group in the intervention period 0.68 0.32–1.42 0.302
Control group in the baseline period 1.63 0.83–3.23 0.160
Control group in the intervention period 1.00 = reference - -
ICU day of sample collection 1.00 0.99–1.02 0.406 0.406
Sample site 0.627
Blood 1.19 0.61–2.36 0.616
Tracheal aspirate 0.95 0.55–1.65 0.858
Urine 0.69 0.36–1.64 0.276
Wound 0.97 0.49–1.95 0.945
Others 1.00 = reference
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Adjusted incidence rate ratios (aIRR) were estimated for all combinations of study groups (chlorhexidine, octenidine, control) and study periods (baseline, intervention) using control group (intervention period) as a reference. ICU day of sample collection, sample sites, cluster effect and interaction between study groups and periods were considered in all models. P-values < 0.05 were interpreted as significant (*). c P-values were reported for comparisons between intervention groups (chlorhexidine and octenidine) and control group (routine care), adjusted by study period. aIRR, adjusted incidence rate ratio. 95%CI, 95% confidence interval.

Discussion

In a large sample of ICU-attributable bacterial isolates collected during the cluster-randomized decolonization trial CLIP-ID, susceptibility to chlorhexidine and octenidine was not reduced 12 months after implementation of daily antiseptic bathing with the respective substances. Some bacterial isolates had enhanced chlorhexidine and octenidine MIC. Most of them were observed in the control group and / or in the baseline period when ICUs did not apply any antiseptic substances. Thus, in our setting, daily bathing with chlorhexidine or octenidine did not enhance the development of non-susceptibilities among bacterial isolates of clinical samples to these substances.

Several in vitro and clinical studies suggest an association of high chlorhexidine use and the development of non-susceptibilities of bacterial isolates to chlorhexidine [17–19, 24, 25]. However, our findings confirmed data from the REDUCE MRSA trial suggesting that chlorhexidine susceptibility of S. aureus isolates was not reduced after implementation of routine chlorhexidine bathing in ICUs [1]. At the same time, data on octenidine susceptibility of bacterial isolates are scarce. Most studies did not report development of non-susceptibility or resistance to octenidine [17, 26–29]. Interestingly, for P. aeruginosa, development of tolerances to both antiseptics, chlorhexidine and octenidine, has been shown in vitro [30]. We observed high chlorhexidine and octenidine MIC for P. aeruginosa. In 90% of P. aeruginosa isolates, chlorhexidine MIC was ≥ 50 μg/ml that represents the species-specific epidemiological cut-off value (Ecoff) as reported by Kampf et al. [18]. However, no P. aeruginosa clinical isolate showed higher MIC than the reference strain.

In our study, the proportions of S. aureus and CoNS isolates with elevated chlorhexidine MIC was high (14.8%; 23 of 155 and 8.2%, 10 of 122) when using MIC ≥ 8μg/ml as Ecoff [1, 18]. This observation, however, was independent from the implementation of antiseptic bathing. For S. aureus isolates, elevated chlorhexidine MIC (32 μg/ml) and octenidine MIC (3 μg/ml) were reported from hospital trusts in the United Kingdom (UK) after high usage of chlorhexidine and implementation of octenidine usage, respectively [16].

Other studies reported lower proportion of S. aureus and CoNS isolates with MIC ≥ 8μg/ml but did not include clinical and / or ICU-attributable isolates [1, 31–33]. In our trial, bacterial isolates were collected from patients who stayed at least 3 days on the ICU. Approximately 70% of ICU patients receive antibiotic therapy [34]. Thus, it is most likely that bacterial isolates from clinical samples in our study were collected from patients previously or currently exposed to antibiotics that might lead to elevated chlorhexidine MIC of their bacterial isolates. However, comparisons to other studies and / or epidemiological cut-off values are only possible to a limited extend. MIC reported here could only be estimated by the concentration of our extraction solutions. Please see limitations for more details.

Our data confirms previous findings that chlorhexidine is highly active against gram-positive and shows lower activity against gram-negative bacteria [35]. Further, estimated MIC required to inactivate bacterial isolates (especially Klebsiella spp. and P. aeruginosa) was higher for chlorhexidine compared with octenidine. These observations confirm in vitro analyses suggesting superiority of octenidine compared with chlorhexidine in the laboratory [36].

The highest estimated chlorhexidine MIC in our study was observed for an E.coli isolate (150 μg/ml) collected during the baseline period. The concentration of chlorhexidine in 2% chlorhexidine-impregnated cloths (= 20.000 μg/ml) is more than 130 times higher. In consequence, the concentration applied to the patient’s skin during antiseptic bathing is expected to be sufficient (20). For octenidine, the highest estimated MIC in this analysis was 40 μg/ml for P. aeruginosa and Klebsiella spp. The octenidine concentration in the 0.08% octenidine (= 800 μg/ml) wash mitts is only 20 times higher, but might still be adequate to reduce the bacterial load on the patient’s skin. Unfortunately, no studies investigating the biocide concentrations that remain on the patient’s skin after bathing with these antiseptic ready-to-use products are available.

Our GEE model found evidence that length of ICU stay (represented by ICU day of sample collection) was associated with higher proportions of bacteria with increased chlorhexidine MIC. Patients with high length of stay on the ICU most likely received more interventions (e.g. invasive procedures, medication, antibiotic therapy) over longer durations of time. As antibiotic resistance is a known indicator of chlorhexidine susceptibility, longer ICU stays (and in consequence presumably more antibiotic treatment) might have an impact on the chlorhexidine susceptibility of bacterial isolates from clinical samples (37). Further, bacterial isolates with high chlorhexidine MIC were significantly less likely to originate from blood cultures compared with other sources. We can only speculate on the reasons for this finding. One possible explanation might be that bacteria isolated from blood might be less affected by ICU interventions including antiseptic bathings compared with other sample sites, e.g. wounds. However, it is unclear, why we did not see these observations with octenidine.

Strengths and limitations

This is one of the largest multicenter studies investigating the susceptibility of bacterial isolates from clinical samples to the antiseptic substances chlorhexidine and octenidine. Bacterial isolates from 60 different ICUs were analyzed, the majority (70.0%) of them sent isolates before and at the end of the intervention period. Further, we included clinical isolates from patients with length of stay on ICU of at least 3 days. Thereby, clinical isolates were collected from patients receiving all treatments applied in the ICU including the respective bathing procedures (chlorhexidine-impregnated cloths, octenidine wash mitts or water and soap) for at least 3 days. Median ICU day of sample collection was 9 days for all species. In consequence, clinical isolates included in this analyses can be designated as ICU-attributable.

Our study has some limitations. First, the number of bacterial isolates tested per species and study group was low. Second, the observation period of 12 months might be too short to draw any final conclusions. In consequence, results must be interpreted with caution. Further, epidemiological cut-off values for chlorhexidine were not available for all species (e.g. CoNS), and not available at all for octenidine (18). Thus, we used species-specific MIC50 as threshold to generate binary variables of phenotypic susceptibilities to chlorhexidine and octenidine for our GEE models. This approach was chosen to apply the same methods for all species and substances (chlorhexidine and octenidine). However, we did not collect all bacterial isolates from clinical samples or a representative sample set from all ICUs participating. Thus, statistical analyses that could be performed to control for potential confounders including cluster-effects were not fully applicable. Fourth, we did not collect any patient-specific data in our analyses (e.g. age, antimicrobial therapy, medication). Potential differences between patient populations of different ICUs could not be considered. Fifth, we did not perform any molecular analyses on the presence of qac genes, efflux pumps, antiseptic resistance genes or any analyses on antimicrobial susceptibility of bacterial isolates. However, this was not subject of our analyses. Several studies are available that investigate the correlation of biocide susceptibility with the absence or presence of efflux pump genes such as qac genes [1, 16, 25, 31, 32]. The correlation of antimicrobial and antiseptic susceptibility is known and has been shown for bacteria such as methicillin-resistant S. aureus (MRSA) and Extended spectrum Beta-Lactamase (ESBL) producing Enterobacterales [31, 37, 38]. Sixth, MIC comparisons to other studies are only possible to a limited extent as no standardized methods for testing phenotypic susceptibility to the antiseptic substances chlorhexidine and octenidine are available [16, 20]. Even though, MIC and minimal bactericidal concentrations (MBC) are most frequently used in this context, no consensus breakpoints for determining susceptibility to chlorhexidine or octenidine exist [16, 20, 25]. Further, MIC tests were not performed with pure chlorhexidine and octenidine substances but with extraction solutions harvested by wringing out the antiseptic products. Pure stock solutions were not provided by the manufacturers. In consequence, MIC reported here could only be estimated from the concentrations reported for the antiseptic products (2μg / ml for chlorhexidine and 0.08 μg/ml of octenidine) and the dilutions (MIC in percentages) derived from of our extraction solutions. This might also limit comparisons to other studies. However, the main focus of our study was whether susceptibilities of bacterial isolates from clinical samples to chlorhexidine and octenidine might decrease from baseline to intervention periods as well as between antiseptic bathing and control group(s). The method of harvesting antiseptic solutions for MIC tests does not have an impact on these results. In fact, using extraction solutions harvested from the antiseptic products is even more close to reality as it included all additional detergents and ingredients from the ready-to-use product that might reduce the properties of pure chlorhexidine and octenidine substances. Further, this approach might consider fractions of chlorhexidine and octenidine remaining in the cloths / mitts during the bathing process. This would not be factored in when using pure stock solutions. Thus, we consider our approach being appropriate and even more conservative. However, our estimated MIC might be overestimated and true MICs of bacterial isolates from clinical samples to chlorhexidine and octenidine might be lower. Finally, MIC of bacterial isolates from clinical samples were determined on ICU- not individual patient level. The longitudinal analyses of bacterial isolates from individual patients before, during and at the end of their ICU stay would allow to directly investigate the potential development of any non-susceptibilities of bacterial isolates from clinical samples to chlorhexidine or octenidine during routine antiseptic bathing. Such elaborate sample collections were conducted for analyses of the skin microbiome including antibiotic resistance genes in this project. Results are currently in preparation. Our study has a high likelihood of being underpowered to show smaller, but also clinically relevant increases in the proportions of non- or less susceptible bacterial isolates from clinical samples (all species) to chlorhexidine or octenidine. However, we discussed limitations in detail and add important data on chlorhexidine and octenidine susceptibilities of bacterial isolates from clinical samples to the scarce literature available on this topic.

Outlook

More analyses on susceptibility of bacterial isolates from clinical samples to antiseptic substances frequently used such as chlorhexidine or octenidine are needed. A standardized protocol would be necessary to compare MIC between different settings and to interpret own findings.

Conclusions

We found no evidence for reduced chlorhexidine or octenidine susceptibilities of bacterial isolates from clinical samples in ICUs after implementation of daily patient bathing with these antiseptics. However, the observation period of 12 months might be too short and the number of bacteria per species and study group too small to draw any final conclusions. In consequence, results must be interpreted with caution.

Supporting information

S1 Data. Data of all bacterial isolates from clinical samples included (n = 790).

(XLSX)

Click here for additional data file. (74.7KB, xlsx)
S1 Table. Reference strains with minimal inhibitory concentrations (μg/ml) to chlorhexidine and octenidine.

(DOCX)

Click here for additional data file. (24.7KB, docx)
S2 Table. Chlorhexidine and octenidine minimal inhibitory concentrations (MIC) of bacterial isolates from clinical samples in percentage of extraction solution.

Median MIC (MIC50) with inter quartile ranges (IQR) and MIC90 are given in in [%] of extraction solution and are reported for Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa stratified by study group (chlorhexidine, octenidine, routine care) and period (baseline = prior to the intervention period, intervention = at the end of the intervention period).

(DOCX)

Click here for additional data file. (32.5KB, docx)
S3 Table. Characteristics of S. aureus isolates from clinical samples with chlorhexidine.

(DOCX)

Click here for additional data file. (28.1KB, docx)
S4 Table. Susceptibility of ICU-attributable bacterial isolates from clinical samples (n = 790) to chlorhexidine and octenidine (all species) stratified by chlorhexidine and octenidine susceptibility.

Chlorhexidine and octenidine susceptibility of bacterial isolates were reported as binary variable (chlorhexidine / octenidine MIC of bacterial isolates ≥ species-specific chlorhexidine/ octenidine MIC50 (yes / no). aP-values were reported for comparisons between chlorhexidine/ octenidine MIC50 ≥ species-specific chlorhexidine/ octenidine MIC50 = “yes” or “no”. b percentage of columns. c percentage of rows. P-values < 0.05 were interpreted as significant (*). n, number. (%), percent.

(DOCX)

Click here for additional data file. (31.2KB, docx)
S1 Checklist. CONSORT 2010 checklist of information to include when reporting a cluster randomised trial.

(DOCX)

Click here for additional data file. (28.8KB, docx)

Acknowledgments

We thank all infection control practitioners, nurses, physicians, healthcare staff and technicians from all ICUs and laboratories participating in and supporting the CLIP-ID trial.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was funded by the German Federal Ministry of Education and Research within the scope of the InfectControl consortium (Grant No. 03ZZ0807A) awarded to PG. Sage Products / Stryker and Schülke, funded the (antiseptic) products for the intervention and supported the investigation of tolerances to chlorhexidine and octenidine by an independent accredited contract laboratory. Dr. Brill + Partner GmbH was the independent contract-laboratory assigned by Schülke and Stryker /Sage to conduct the investigations of tolerances to chlorhexidine and octenidine (by minimum inhibitory concentration testing). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Surbhi Leekha

12 Sep 2022

PONE-D-22-18018Chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples in a three-armed cluster randomised decolonisation trialPLOS ONE

Dear Dr. Denkel,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Surbhi Leekha

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

   "We thank all infection control practitioners, nurses, physicians, healthcare staff and technicians from all ICUs and laboratories participating in and supporting the CLIP-ID trial. We are grateful to Sage Products 17/ Stryker and Schülke as manufacturers of the (antiseptic) products applied in this trial, for their support. We thank Dr. Brill + Partner GmbH, Hamburg, Germany for their contract work."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

 "Funding for the CLIP-ID trial was provided by the German Federal Ministry of Education and Research within the scope of the InfectControl consortium (03ZZ0807A). Sage Products / Stryker and Schülke funded the (antiseptic) products for the intervention and supported the investigation of tolerances to chlorhexidine and octenidine by an independent accredited contract laboratory. The German Ministry of Education and Research (funder) and companies (Sage Products / Stryker, Schülke) funding products and the investigation of tolerances to chlorhexidine and octenidine by an independent laboratory had no role in study design, data collection and analysis, data interpretation, the decision to publish, or the preparation of the manuscript."

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Additional Editor Comments:

The manuscript was reviewed by two reviewers with expertise in this area and a statistical reviewer. Please note their comments below with which I agree. Specifically,

1. Please provide the rationale for use of and methods used to determine of the concentration of antiseptic solution generated from wringing out wipes (vs using stock solution)

2. As pointed out by the statistical reviewer, provide power calculations on the effect size for differences in antiseptic susceptibility that could be detected with the target sample size that was already set based on the primary clinical outcome of the trial. The use of more appropriate statistical models is also suggested to statistically compare differences between groups over time.

Minor:

Please clarify what the proportions of different bacterial species shown in Table 2 represent, i.e., what is the denominator?

Please correct the following sentence in the conclusion: “However, the observation period of 12 months might be too and the number of bacteria per species and study group too short to draw any final conclusions.”

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall, I find this paper interesting with large pool of isolates and interesting set of data which may be used for other researches. I believe this paper should be published, followed some clarifications.

My comments are listed below:

Comment 1: The methods are unclear

1. It is unclear how did you determine the antiseptic stock solutions concentrations of antiseptic substances (chlorhexidine and octenidine), given the fact that they were 'harvested by wringing out'.

2. the production of the Bacterial inoculum is also unclear. It seems that the range of the bacteria CFU is to high 1.5-5.0 108, is it spelling mistake?

Did you meant that the bacterial suspensions were diluted to a final concentration of 10-6 and 10-7? if so, it's need to be better clarify. furthermore, is the bacterial concentration calculated here is before or after the antiseptic and the CSL mixing?

Comment 2: You have a lot of information regarding the isolate origin- e.g blood, urine, tracheal aspirate and liquor- did you looked into differences base on that? If so, show it. It's an interesting data that may contribute largely to current knowledge.

Reviewer #2: The authors performed a study of phenotypic chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples obtained before and one year after implementation of a three-armed cluster randomized decolonization trial. The results add to the existing literature addressing the impact of antiseptics on resistance patterns. The study design utilized clinical samples and called for the comparison of bacterial isolates obtained prior to initiation of the study with those obtained at the end of the study. This design results in a population-level comparison and the authors acknowledge the limitations of this type of design compared to that of longitudinal patient sampling. The authors also recognize in detail other limitations of their design in the Discussion. Further, the authors inform the readers that a longitudinal patient-level collection of bacterial isolates was done to study the cutaneous microbiome over time, although it’s unclear whether that study will result in comparisons of resistance markers over time in the three arms. It’s also the case that bacteria found in tracheal aspirates, urine, and CSF may have less of an opportunity to be affected by antiseptics used on the skin. That said, the authors provide the results by bacterial species and clinical sample type, which is a strength of their presentation.

Specific Comments/Questions.

1. Abstract, 3rd paragraph of Introduction. Rather than using the terminology “side effects”, something like “unintended consequences” might be a better choice.

2. Methods. Why were stock solutions created by wringing out the impregnated cloths and wash mitts rather than purchasing stock solutions or creating stock solutions from a more concentrated liquid source?

Reviewer #3: A 3-arm cluster randomized clinical trial was conducted which aimed to assess the phenotypic susceptibility of bacterial isolates to chlorhexidine or octenidine for antiseptic bathing. The conclusions are unclear.

Major revision:

More sophisticated statistical models to test for group by time interactions are called for.

Minor revisions:

1- Table 1, title: In statistical terms, stratification refers to a process used during randomization. Consider replacing this term in the title.

2- State and justify the study’s target sample size with a pre-study statistical power calculation. The power calculation should include: sample size, alpha level (indicating one or two-sided), minimal detectable difference and statistical testing method.

3- To assist in the review process, add line numbering to the document.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2022 Dec 14;17(12):e0278569. doi: 10.1371/journal.pone.0278569.r002

Author response to Decision Letter 0

27 Oct 2022

Dear editor, dear reviewers,

Thank you very much for your thorough and helpful comments and advises on our manuscript PONE-D-22-18018 entitled “Chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples in a three-armed cluster randomised decolonisation trial”. Please find our point by point response in the cover letter entitled PONE_D-22-18018_R1_Rebuttal_final.docx" that we uploaded as additional file. All lines reported in the rebuttal letter refer to the manuscript with track changes.

Comments by editor and reviewers are marked in grey with black font, responses by the authors in blue fonts without marks and changes in the manuscript were marked in yellow with black font.

We hope that we now fulfill all journal requirements, that we could answer all open questions to your full satisfaction and that we could dissolve all your concerns. Please do not hesitate to contact us, if you need any further information or have any further questions. Thank you very much in advance.

Kind Regards,

Luisa Denkel

Attachment

Submitted filename: PONE_D-22-18018_R1_Rebuttal_final.docx

Click here for additional data file. (44.2KB, docx)

Decision Letter 1

Surbhi Leekha

21 Nov 2022

Chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples in a three-armed cluster randomized decolonization trial

PONE-D-22-18018R1

Dear Dr. Denkel,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Surbhi Leekha

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

Acceptance letter

Surbhi Leekha

1 Dec 2022

PONE-D-22-18018R1

Chlorhexidine and octenidine susceptibility of bacterial isolates from clinical samples in a three-armed cluster randomised decolonisation trial

Dear Dr. Denkel:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Surbhi Leekha

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Data. Data of all bacterial isolates from clinical samples included (n = 790).

    (XLSX)

    Click here for additional data file. (74.7KB, xlsx)
    S1 Table. Reference strains with minimal inhibitory concentrations (μg/ml) to chlorhexidine and octenidine.

    (DOCX)

    Click here for additional data file. (24.7KB, docx)
    S2 Table. Chlorhexidine and octenidine minimal inhibitory concentrations (MIC) of bacterial isolates from clinical samples in percentage of extraction solution.

    Median MIC (MIC50) with inter quartile ranges (IQR) and MIC90 are given in in [%] of extraction solution and are reported for Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa stratified by study group (chlorhexidine, octenidine, routine care) and period (baseline = prior to the intervention period, intervention = at the end of the intervention period).

    (DOCX)

    Click here for additional data file. (32.5KB, docx)
    S3 Table. Characteristics of S. aureus isolates from clinical samples with chlorhexidine.

    (DOCX)

    Click here for additional data file. (28.1KB, docx)
    S4 Table. Susceptibility of ICU-attributable bacterial isolates from clinical samples (n = 790) to chlorhexidine and octenidine (all species) stratified by chlorhexidine and octenidine susceptibility.

    Chlorhexidine and octenidine susceptibility of bacterial isolates were reported as binary variable (chlorhexidine / octenidine MIC of bacterial isolates ≥ species-specific chlorhexidine/ octenidine MIC50 (yes / no). aP-values were reported for comparisons between chlorhexidine/ octenidine MIC50 ≥ species-specific chlorhexidine/ octenidine MIC50 = “yes” or “no”. b percentage of columns. c percentage of rows. P-values < 0.05 were interpreted as significant (*). n, number. (%), percent.

    (DOCX)

    Click here for additional data file. (31.2KB, docx)
    S1 Checklist. CONSORT 2010 checklist of information to include when reporting a cluster randomised trial.

    (DOCX)

    Click here for additional data file. (28.8KB, docx)
    Attachment

    Submitted filename: PONE_D-22-18018_R1_Rebuttal_final.docx

    Click here for additional data file. (44.2KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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