Acquired resistance to chlorhexidine – is it time to establish an ‘antiseptic stewardship’ initiative?

Accepted Manuscript Acquired resistance to chlorhexidine – is it time to establish an “antiseptic stewardship” initiative? Günter Kampf PII:

S0195-6701(16)30374-7

DOI:

10.1016/j.jhin.2016.08.018

Reference:

YJHIN 4901

To appear in:

Journal of Hospital Infection

Received Date: 27 July 2016 Accepted Date: 18 August 2016

Please cite this article as: Kampf G, Acquired resistance to chlorhexidine – is it time to establish an “antiseptic stewardship” initiative?, Journal of Hospital Infection (2016), doi: 10.1016/j.jhin.2016.08.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Acquired resistance to chlorhexidine – is it time to establish an “antiseptic stewardship” initiative?

Günter Kampf 1,2*

und Team GmbH, Infection Control Science, Kattrepelsbrücke 1, 20095 Hamburg, Germany; email: [email protected]; tel. +49 40 328907275 2Ernst-Moritz-Arndt

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1Knieler

Universität, Institut für Hygiene und Umweltmedizin, Walter-RathenauStraße 49 A, 17475 Greifswald, Germany

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Running title: chlorhexidine use and acquired resistance

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Key words: chlorhexidine, resistance, cross resistance, exposure, selection pressure, antiseptic stewardship

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ACCEPTED MANUSCRIPT Summary

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Chlorhexidine digluconate (CHG) is an antimicrobial agent used for different types of applications in hand hygiene, skin antisepsis, oral care and patient washing. Increasing use raises concern on a development of acquired bacterial resistance. Published data from clinical isolates with CHG MICs were reviewed and compared to epidemiological cut-off values to determine resistance. CHG resistance is rarely found in E. coli, Salmonella spp., S. aureus and CNS. In Enterobacter spp., Pseudomonas spp., Proteus spp., Providencia spp. and Enterococcus spp., however, isolates are more often CHG resistant. CHG resistance can be detected in multiresistant isolates such as XDR K. pneumoniae. Isolates with a higher MIC are often less susceptible to CHG for disinfection. Although cross-resistance to antibiotics remains controversial some studies indicate that the overall exposure to CHG increases the risk for resistance to some antibiotic agents. Resistance to CHG has resulted in numerous outbreaks and healthcare-associated infections. On an average intensive care unit most of the CHG exposure would be explained by hand hygiene agents when liquid soaps or alcohol-based hand rubs contain CHG. Exposure to sublethal CHG concentration can enhance resistance in Acinetobacter spp., K. pneumoniae and Pseudomonas spp., all species well known for emerging antibiotic resistance. In order to reduce additional selection pressure in nosocomial pathogens it seems to make sense to restrict the valuable agent CHG to those indications with a clear patient benefit and to eliminate it from applications without any benefit or with a doubtful benefit.

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ACCEPTED MANUSCRIPT Introduction

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Chlorhexidine digluconate (CHG) was first synthesized in the fifties in Great Britain when a broad screening exercise to find active agents against malaria was performed.1 The active agent is now used for a variety of applications with some of them being justified by strong scientific evidence. One of them is the use as an antiseptic rinse for the oral cavity in ventilated patients with the aim to prevent ventilator-associated pneumonia.2 Another one is in combination with alcohols the treatment of puncture sites of central venous catheters with the aim to reduce catheter-associated bloodstream infections.3,4

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Some other applications, however, are disputable, e.g. its routine use for washing patients on intensive care units,5,6 its use in combination with alcohols for the pre-operative treatment of skin,7 and its use in hand hygiene.8 In a good number of countries liquid soaps based on 4% CHG are used for hygienic or surgical hand wash. CHG can also be found in alcohol-based hand rubs, e.g. in a formulation with 70% iso-propanol and 0.5% CHG which was used in an internationally respected study on the effect of compliance in hand hygiene on the incidence of healthcareassociated infections. 9

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The overall risk for an acquired resistance to biocidal agents such as CHG is considered to be small as long as the antiseptics are used correctly.10 At the same time a number of outbreaks has been reported associated with contaminated CHG solutions.11 These studies indicate the strong ability of microorganisms to adapt to CHG. Already in 1973 a “disinfectant failure” was described in the context of CHG-containing disinfectants because some Gram-negative bacterial species such as Pseudomonas or Klebsiella were able to withstand the treatment.12 The wider use of CHG should therefore also include an evaluation of the potential of an emerging microbial CHG resistance.13

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One mechanism of CHG resistance is efflux pumps which can pump out of the bacterial cell various types of antibiotics and biocidal agents including CHG.14 Among 114 effluxing Staphylococcus aureus isolates CHG was effluxed in 96% of the strains indicating the potential of efflux pumps.15 Other mechanisms of resistance are the inactivation of the active ingredient or changes of the cell wall structure.16 Aquatic environmental isolates of Gram-negative species, e.g. from treated wastewater, are particularly often resistant to CHG.17 Resistance to biocidal agents and antibiotics is often mediated by the same plasmids.18,19 That is why it seems necessary to address the issue of CHG resistance.

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Epidemiological cut-off values to determine resistance to CHG The greater use of CHG warrants surveillance for resistance.20 In order to identify biocide resistance, it has been proposed by Morrissey et al to base the definition of resistance on the “natural” susceptibility to antimicrobials of a given species and not just on the clinical success of the treatment when a microorganism is defined as resistant by a level of antimicrobial activity associated with a high likelihood of therapeutic failure.21 A wild type isolate has a “natural” susceptibility to antimicrobials in the absence of acquired and mutational mechanisms of resistance to the agent.21 The analysis of the wild type MIC phenotype of several unrelated isolates allows establishing the epidemiological cut-off value which is the upper limit of the normal MIC distribution for a given antimicrobial and a given species.21 Any isolate presenting a MIC above this value is considered as “resistant” irrespective of whether or not the achieved level of resistance comprises therapy or antimicrobial efficacy in clinical medicine.21 It is Page 3

ACCEPTED MANUSCRIPT possible that these MIC values bear no clinical relevance but there are certainly helpful to identify acquired resistance to an agent and quantify the dimension. In 2014 epidemiological cut-off points were proposed to classify isolates as CHG susceptible or resistant for various bacterial species and C. albicans, based on an analysis of 3227 clinical isolates and their MIC values (Table I).21 In 1982 it was proposed for P. aeruginosa to classify isolates with a CHG MIC value of at least 50 mg/l as resistant based on a bimodal distribution curve of susceptibility.22.The same cut-off value was suggested for mycobacteria.23

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Susceptibility to CHG in relation to epidemiological cut-off values

All data summarized below describe the range and median MIC values for various nosocomial pathogens obtained with CHG in relation to the epidemiological cut off values. But it should be kept in mind that a resistance based on epidemiological cut-off values is not automatically evidence for resistance in clinical applications.24

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Escherichia coli

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Based on the data from 4 studies (Table I) and considering the proposed epidemiological cut-off value (64 mg/l) one can see that E. coli isolates that would be considered CHG resistant are rare. After repetitive exposure to sublethal concentrations of CHG no relevant MIC increase was found (≤ 0.7 mg/l).25 Out of 374 clinical isolates from patients with a urinary tract infection most isolates were found with MIC values ≤ 10 mg/l, and no isolate was identified with an MIC value MHK > 500 mg/l.26,27 Isolates obtained from urine after repetitive CHG bladder washout could still be completely killed by 500 mg/l CHG within 15 min.28 Klebsiella spp.

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Already in 1980 some highly resistant isolates (1.8%) with a CHG MIC value > 500 mg/l were reported out of a total of 167 Klebsiella isolates from patients with a urinary tract infection.26 In the majority of studies maximum MIC values were found beyond the epidemiological cut off value of 64 mg/l although the median MIC values were still below 64 mg/l in 3 of the four studies (Table I). Only among the 126 XDR-isolates from Israel a high mean of 140 mg/l indicates that the magnitude of antibiotic resistance correlates with the magnitude of CHG resistance.

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Data from other studies contribute to the picture. After repetitive bladder washout (100 ml of a CHG solution with 200 mg/l) in patients with a transurethral catheter K. pneumoniae isolates with MIC values of 40 mg/l were found.29 A pan-resistant K. pneumoniae isolate could even multiply in a liquid soap containing 1% CHG.30 New data indicate that K. pneumoniae strains can adapt quite easily to CHG resulting in a 128-fold decrease of susceptibility to CHG.31 The reduced susceptibility to CHG and the frequent detection of various resistance genes was regarded as an indicator for a strong exposure to CHG and a strong distribution of CHG resistance.32 The clinical relevance of elevated MIC values in Klebsiella is not entirely clear. K. pneumoniae isolates with a MIC value of 200 mg/l can mostly be killed in 10 min by 500 mg/l CHG (mean log10-reductions between 4.2 and 5.8).28 The efficacy of hand hygiene preparations within 1 min, however, was significantly impaired against adapted isolates.31 Enterobacter spp In 8 clinical Enterobacter spp. strains the CHG MIC values were between 10 – 75 mg/l.33 New data from Mexico show a 100% resistance rate that among 24 clinical isolates of ESBL E. cloacae mostly encoded by the resistance gene qacE delta1.34 Page 4

ACCEPTED MANUSCRIPT Considering the epidemiological cut off value of 16 mg/l it can be concluded that a large number of Enterobacter spp. isolates can be classified as resistant to CHG. Due to a lack of data with Enterobacter spp. it is, however, not clear if these isolates are more difficult to kill with higher CHG concentrations. Salmonella spp.

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One study addressed the CHG susceptibility of 195 Salmonella spp. isolates from chicken and egg production chains (Table I). The range was from < 4 to 64 mg/l with a median of 16 mg/l.35 Taking into account the proposed cut-off value to determine CHG resistance (32 mg/l) the majority of isolates can be considered susceptible. Pseudomonas spp.

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The range of MIC values from clinical isolates and reference strains of Pseudomonas spp., P. aeruginosa and P. stutzeri are summarized in Table I. Most studies show CHG MIC values beyond or even far beyond the cut off value which has been proposed to determine resistance in P. aeruginosa. Data from 14 additional clinical isolates from patients with a urinary tract infection show that 35.7% of them were highly resistant with MIC values> 500 mg/l.27 After repetitive bladder washouts using 100 ml of a CHG solution with 200 mg/l among patients with a transurethral catheter P. aeruginosa was isolated with CHG MIC values of 80 mg/l.29 Pseudomonas spp. was also detected as a contaminant in CHG solutions of unknown strength in 11 out of 120 samples (9.2%); the solutions were used in pediatrics, neonatology and surgery.36 A multi-resistant isolate of P. aeruginosa was even able to multiply in a liquid soap containing 1% CHG.30Isolates with a very high MIC to CHG (e.g. 800 mg/l) were found to have only a very reduced susceptibility to CHG at 500 mg/l so that in an exposure time of 10 to 60 min only a log10-reduction of 2.0 to 3.1 can be found.28

Providencia spp.

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Pseudomonas spp. can adapt or develop resistance to CHG quite easily.37 After repetitive exposure to sublethal concentrations of CHG a substantial increase of the CHG MIC was observed from 10 mg/l to 70 mg/l.25 Sublethal CHG concentrations can activate efflux pumps such as the MexCD-Op which can eliminate antibiotics out of the bacterial cell.38-40 Changes of the outer membrane were detected in CHG resistant cells (MIC of 100 mg/l) in P. stutzeri.41,42 Biofilm may also play a role because CHG at 4% was found to have a particularly poor efficacy against P. aeruginosa when present in biofilm.43

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Resistance to CHG in Providencia spp. was described frequently especially in isolates from patients with a urinary tract infection. In one study 83.3% of the 24 P. stuartii isolates were highly resistant to CHG with an MIC > 500 mg/l.26 Another study revealed a proportion of 68.7% of P. stuartii isolates with a MIC > 500 mg/l among 16 clinical isolates.27 A third study described that out of 70 Providencia spp. isolates most of them had an MIC of 1600 mg/l, followed by 800 mg/l.44 Repetitive bladder washouts with 100 ml of a solution with 200 mg/l CHG in patients with a transurethral urinary catheter resulted in isolates of P. stuartii with MIC values of 640 mg/l.29 Resistance to CHG in P. stuartii is probably mediated by the inner membrane.45 Providencia spp. isolates with a MIC value of 500 mg/l cannot be killed anymore by CHG in 500 mg/l, the log10-reduction after 60 min is only between 0.2 and 0.6.28 Acinetobacter spp.

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ACCEPTED MANUSCRIPT Most studies show low MIC values for CHG in Acinetobacter spp. (Table I). To what extent a higher MIC value in Acinetobacter spp. changes the efficacy of CHG in antiseptics remains debatable due to a lack of data.

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Exposure of A. baylyi ADP1 to sublethal concentrations of CHG induced a reduced susceptibility against lethal concentrations of CHG.46 Repetitive use of CHG as a mouth rinse and in liquid soaps for washing patients on various departments changed the susceptibility in XDR A. baumannii. The MIC50 before the intervention was between 16 and 32 mg/l; after the intervention it had increased to 64 mg/l.47 A pan-resistant isolate of A. baumannii was even able to multiply in a liquid soap based on 1% CHG.30 A mechanism of resistance in Acinetobacter spp. are efflux pumps such as AceI.48 Serratia spp.

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Reference strains of S. marcescens usually show MIC values between 2.5 and 25 mg/l (Table I). The analysis of 131 strains obtained from patients with a urinary tract infection showed MIC values between 3 and 200 mg/l. In one of the four serotypes the MIC50 was high with 100 mg/l (Table I). To what extent a higher MIC value in Serratia spp. changes the efficacy of CHG in antiseptics remains debatable due to a lack of data.

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An ESBL S. marcescens was able to persist and continuously contaminate a CHG stock solution of an unknown concentration in a Mexican children’s hospital and may have contributed to an outbreak involving 20 patients.49 Repetitive exposure of Serratia spp. to CHG resulted in a stable resistance to CHG.50 Various mechanisms of resistance have been described. A change of the inner cell membrane has been described, as well as efflux pumps.51,52 S. marcescens cells resistant to CHG also have a wrinkled outer cell membrane and produce an additional protein with an unknown function.53

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Proteus spp.

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All data from clinical isolates and reference strains are summarized in Table I. Most studies indicate high MIC values for clinical isolates of Proteus spp. Without knowing the epidemiological cut-off value to determine resistance in Proteus spp., however, it remains difficult to describe the proportion of resistant isolates among all clinical isolates. Highly resistant isolates of P. mirabilis (MIC value of 1280 mg/l) were identified after repetitive bladder washouts with 100 ml of a solution of 200 mg CHG / ml in patients with a transurethral catheter 29. Even isolates with an MIC value of 800 mg/l still have some susceptibility to CHG at 500 mg/l when a log10-reduction of 4.0 after 15 min or > 6 after 30 min can be found.28 Staphylococcus aureus

An overview of MIC values obtained in clinical isolates of S. aureus is provided in Table I. Although the MIC may be higher than 8 mg/l the medians indicate that the majority of clinical S. aureus isolates can be considered to be susceptible to CHG. Other studies provide evidence that MRSA is less susceptible to CHG compared to MSSA. In 1996 it was described that the mean MIC value for MRSA is five to ten times higher compared to MSSA.54 This difference was confirmed in other studies. In a study from Nigeria 41 isolates were analyzed with an MIC50 of 2 mg/l for the 25 MSSA isolates and of 32 mg/l for the 16 MRSA isolates.55 From 4 hospitals in Iran it was reported that 30% of 100 MSSA isolates have an MIC between 8 and 16 mg whereas the rate was 70% in 100 MRSA isolates.56 A higher MIC value to CHG in S. aureus, however, does not necessarily mean an impaired efficacy in clinical applications against these isolates or strains.57,58 Page 6

ACCEPTED MANUSCRIPT After 20 years of using a 4% CHG liquid soap in Taiwan it was observed that the proportion of MRSA isolates with an MIC value ≥ 4 mg/l increased from 1.7% in 1990 to 50% in 1995, 40% in 2000 and 46.7% in 2005.59 A reduced susceptibility to CHG is associated with the qacC gene. In 69 S. aureus isolates collected in 14 months from wound swabs the qacC gene was detected in 18.8% with eleven of them with a MIC value ≥ 1 mg/l.60 An adaptation in S. aureus to CHG is rather unlikely. One S. aureus isolate did not show an increase of the MIC after 100 days of exposure to CHG at a concentration half of the MIC.61

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Coagulase-negative Staphylococcus spp. (CNS) Data from three studies were evaluated (Table I), the majority of isolates showed low MIC values to CHG. Enterococcus spp.

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An analysis of clinical isolates of E. faecalis from Estonia VRE isolates from the UK revealed low MIC values (Table I). In a large survey with 107 E. faecalis and 165 E. faecium isolates from various sources including clinical material, however, the MIC values were found to be between 2.5 and 2500 mg/l. A noticeable finding was that 74.6% of the highly resistant isolates (2500 mg/l) came from clinical material (Table I). Taking into account the proposed cut-off values for CHG resistance (E. faecium: 32 mg/l, E. faecalis: 64 mg/l) it is striking that resistant isolates are mainly found in clinical material. To what extent a higher MIC value in Enterococcus spp. changes the efficacy of CHG in antiseptics remains debatable due to a lack of data. Similar to other bacterial species resistance to CHG in enterococcus spp. is often mediated by efflux pumps such as EfrAB.62 Common resistance genes

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qacA/B gene

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Multidrug efflux pumps can be divided into 5 protein families depending on their energy requirements and structure.63 Two of them are the ‘Major Facilitator Superfamily’ (MFS) and the ‘Small Multidrug Resistance’ (SMR) family.63 MFS represents the largest known family of secondary transporter systems with at least 74 protein families including qacA and qacB.63 Qac genes (‘quaternary ammonium compound’), also described as biocide or antiseptic resistance genes, 64,65 are most commonly found in isolates suspected to be CHG resistant. Detection of the qac gene is associated with a significantly higher MBC for 4% CHG which was determined in 94 S. aureus isolates with 38 of them being HA-MRSA, 25 CA-MRSA, 6 VISA and 25 MSSA.65

The qacA/B gene confers to CHG resistance;58 it is found in S. aureus and coagulase-negative staphylococci in the chromosome,66,67 and on plasmids including the pSK1-plasmid family.66 qacB transfers in S. aureus a resistance to monovalent organic cations and in addition on a low level to some bivalent substances.66 It can be found on various plasmids such as β-lactamase and on heavy metal resistance plasmids (pSK23).66 qacA and qacB are very similar and difficult to distinguish by PCR.66 Many isolates are present with highly polymorphic forms of these genes. The functional differences between qacA and qacB originally reported by Paulsen et al are now less clear.68 qacA/B is considered to be the most frequent resistance gene for biocidal agents in disinfectants.69 MRSA strains carrying the qacA/B gene have been described to have a CHG MIC value of 256 mg/l in the presence of 3% bovine serum albumin.70 In combination with low-level resistance to mupirocin the presence of qacA/B in MRSA significantly increases the risk of persistent MRSA carriage after decolonization therapy.71 qacA/B- and smr-positive S. aureus isolates were more often associated with invasive bloodstream infections.72 The authors Page 7

ACCEPTED MANUSCRIPT suggested that this may be reflective of the use of CHG in the cleansing and maintenance of CVLs and the subsequent selection of antiseptic-tolerant organisms.72 It has been claimed that the chronological emergence of qacA and qacB determinants in clinical isolates of S. aureus mirrors the introduction and usage of cationic biocides.73 Table II provides an overview on the frequency of the qacA/B resistance genes in MRSA.

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The qacA/B gene can be found in 2.1% to 80% of clinical MRSA isolates (Table II). In MSSA the detection rates are lower with 2% to 12%.66,74,75 The trend in MRSA is increasing. In a pediatric oncology unit in the US the qacA/B rate among MRSA increased year by year.76 The qacA gene can also be found in gram-negative species. Among 27 clinical isolates of carbapenem-resistant K. pneumoniae 41% were qacA carrier.77 Detection of the qac gene correlated with a reduced susceptibility to biocidal agents such as chlorhexidine acetate.77

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qacE gene

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The qacE gene was first detected in E. coli.63 A functional deletion variant exists (‘qacEΔ’), which can also be found in other species. Both can be found quite commonly in Gram-negative species. In 27 clinical isolates of carbapenem-resistant K. pneumoniae qacE was found in 15% and qacEΔ in 59%.77 Detection of qacE or qacEΔ correlated with a reduced susceptibility to biocidal agents including chlorhexidine acetate.77 In 122 multi-resistant A. baumannii isolates in Malaysia qacE was detected in 73%. The MIC values for CHG, however, were all between 0.2 and 0.6 mg/l indicating phenotypical susceptibility to CHG.78 Finally, a study on 64 K. pneumoniae isolates from Scotland found qacEΔ in 34 of them and qacE in one of them.32 Smr gene

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The smr gene was first detected on a S. aureus plasmid and describes ‘staphylococcal multidrug resistance’.63 It turned out to be identical with the qacC gene.63 Irrespective of its description it belongs to the smr protein family and is also regarded as a biocide or antiseptic resistance gene.70,79 Today, both descriptions (smr gene and qacC gene) are used synonymously.63

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In Korea the smr gene was detected in 71% among 456 MRSA isolates.80 A study in various Asian countries with a total of 894 MRSA isolates indicated an over presence of smr in 3.1% of the isolates with a significantly higher rate (31.6%) in India.81 In a study from Japan, the smr detection rate in 283 MRSA isolates was 1.4%;82 in another one it was 3.3% in 334 MRSA isolates and 5.9% in 188 MSSA isolates.75 Data from Canada revealed a rate of 6.9% in 334 MRSA isolates.83 From Shanghai in China a rate of 77.4% was reported in 53 MRSA isolates.70 Most of the isolates were not susceptible to CHG when exposed under high organic load (3% bovine serum albumin), a mean MBC of 256 mg/l was described.70 In Scotland smr was detected in 44.2% among 120 MRSA isolates.84 qacC was found in 6 of 61 coagulase-negative staphylococcus isolates with 5 of them being resistant to CHG.60 In MRSA presence of the smr gene is associated with a phenotypically reduced susceptibility to CHG. In one study the MBC to CHG was determined in 88 MRSA isolates. Whenever the MBC was 5 mg/l smr was present in 15% of the isolates. In isolates with a MBC of 10 mg/l the proportion was 28%, and in isolates with a MBC of 20 mg/l the proportion was even 50%.83 In another study with 400 isolates of S. aureus a similar finding was reported. Whenever the smr gene was present the mean MBC was 16 mg/l. In isolates without the smr gene the mean MBC was 2 mg/l.85

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ACCEPTED MANUSCRIPT CHG cross-resistance to antibiotics A possible cross-resistance between CHG and antibiotics is controversial.86,87 The widespread use of CHG has not yet resulted in a clinically relevant resistance to antibiotics,88,89 even though development of resistance to these agents is regarded as realistic 73. Such a development is more likely to occur in clinical medicine, rather than in industry, because the selection pressure by these substances is much higher in patient care.90

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Some studies have described that there is no cross resistance between CHG and antibiotics. Among 101 genetically distinct isolates of the B. cepacia complex no correlation was found between the susceptibility to CHG and 10 different antibiotics.91 In 130 Salmonella spp. from two turkey farms no cross resistance between CHG and five antibiotics was found.92

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Other studies indicate that cross resistance between CHG and antibiotics does occur. An analysis of 701 Gram negative strains in 1991, representing 16 species or bacterial genera, showed that there was a positive correlation between resistance to antiseptics (cetrimide, chlorhexidine, hexachlorophene) and to antibiotics for S. marcescens and Alcaligenes spp.93 In 49 A. baumannii strains with a reduced susceptibility to CHG a co-resistance to carbapenem, aminoglycoside, tetracyclin and ciprofloxacin was found.94 In Trinidad 11 of 120 CHG solutions were found to be contaminated with Pseudomonas spp., with resistance rates to ciprofloxacin of 58.3%, to norfloxacin of 50.0%, to tobramycin of 45.8%, and to gentamicin with 41.7%.36 In a CHG resistant Pseudomonas stutzeri isolate a cross-resistance to polymyxin and gentamicin was found.41 It is also remarkable that the highest median MIC values for CHG were reported in XDR K. pneumoniae (Figure 1), especially since in 2014 a multidrug efflux pump was detected in K. pneumoniae which can eliminate a variety of antibiotics and biocidal agents out of the bacterial cell.95 kpnEF is one SMR-type efflux pump in K. pneumoniae which is directly involved in capsule formation causing hypermucoviscosity.96 In addition, it may cause resistance to some antibiotics such as cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin, and to some biocidal agents such as benzalkonium chloride, chlorhexidine, and triclosan.96 In Japan an outbreak of 7 cases of catheter-associated urinary tract infection caused by multiresistant P. aeruginosa was analyzed. The outbreak strain was resistant to CHG and at the same time resistant to 25 of 27 tested antibiotics whereas a CHG resistant ATCC strain did not show a resistance to the antibiotics.97 An analysis of 148 E. coli clinical isolates from clinical lesions showed that 12.8% were classified as resistant to CHG (MIC ≥ 5 mg/l), and they were also multiply drug resistant and multiply metal resistant.98 Exposure of Burkholderia spp. to 0.005% CHG for 5 min resulted in a significant reduction of susceptibility to ceftazidime, ciprofloxacin and imipenem in 2 of 4 experiments although a clinical interpretation was not possible for the authors.99 Cross resistance has also been described in Gram-positive species. When healthcare workers used a soap based on 2% CHG they had a relative risk of 1.92 to be colonized on their hand with a S. epidermidis resistant to oxacillin and 1.50 for resistance to gentamicin. In S. warneri the relative risk for rifampicin resistance was even 7.22.100 An analysis of 301 S. aureus isolates from three African countries showed a significant association between specific resistance genes for biocidal agents (sepA, mepA, norA, lmrS, qacAB, smr) and resistance to antibiotics.101 Recent data show that exposure of vancomycin-resistant E. faecium to CHG for only 15 min upregulates the vanA-type vancomycin resistance gene (vanHAX) and genes associated with reduced daptomycin susceptibility (liaXYZ).102 In another study 120 clinical MRSA isolates were exposed to various concentrations of CHG (range: 2.5 – 40 mg/ml) which was allowed to dry in a glass bottle. Changes in the susceptibility to eight antibiotics (ampicillin, tetracycline, vancomycin, gentamicin, oxacillin, cefotaxime, cefuroxime, ciprofloxacin) was determined. MICs of Page 9

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cefotaxime, vancomycin, gentamicin, cefuroxime and oxacillin increased in EMRSA-16 following 48 h of residue drying. There were also increases in the MICs of all tested antibiotics for the NCTC 6571, a S. aureus susceptible strain, following exposure to chlorhexidine residues that had been drying for 48 h (compared with the MICs for the strain before exposure). The increases in the MICs of all tested antibiotics for the susceptible control S. aureus strain following exposure to surface dried chlorhexidine residues is of interest as it suggests that the use of chlorhexidine in the hospital environment may be linked to increased resistance to antibiotics in previously susceptible strains.84 An analysis of 247 nosocomial S. aureus isolates revealed that smr-positive S. aureus isolates (44.0%) were more often resistant to methicillin, ciprofloxacin, and/or clindamycin.72 The isolates positive for qacA/B (33.6%) had more often a vancomycin MIC of ≥ 2 μg/ml.72 An analysis of multiresistance plasmids found in 280 staphylococcal isolates from diverse geographical regions from the 1940s to the 2000s suggested that enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.103

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Transfer of resistance genes

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Studies of resistance to antimicrobials have revealed that resistance genes are probably moving to plasmids from chromosomes more rapidly than in the past and resistance genes are aggregating upon plasmids.104 Some resistance genes may be transferred between bacterial species. The qacA-gene, for example, is often carried on a plasmid from the pSK1 family of vector,105,106 but other plasmids may also carry the resistance gene and can be transferred.20 Another example is the plasmid pTZ2162qacB which was able to transfer the qacB-gene horizontally to MRSA by transduction.107 Clinical impact of acquired CHG resistance

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Various reports of outbreaks caused by contaminated CHG solutions, mostly at 0.05%, are described for different bacterial species such as Pseudomonas spp., P. aeruginosa, S. marcescens, B. cepacia, R. picketti and A. xylosoxidans.11 In addition, some outbreaks have been reported which were attributed to an acquired resistance to CHG. They are summarized in Table III. CHG exposure in healthcare

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In the UK the MIC values of 251 clinical isolates to CHG were opposed to the magnitude of CHG exposure from different types of antiseptics (CHG in water, soap or alcohol solutions). A clear correlation between the exposure and the mean MIC was found. In isolates obtained from patients with low exposure the mean MIC was 10 mg/l, in moderate exposure it was 15 mg/l, and in high exposure it was 25 mg/l.108 When different bacterial species (S. aureus, E. coli, P. aeruginosa, S. marcescens, C. albicans) are exposed to CHG for 12 weeks the MIC increases substantially, e.g. 4-fold in C. albicans, 16-fold in S. aureus, 32-fold in E. coli and P. aeruginosa, and 128-fold in S. marcescens.109 An exposure for up to 24 h, however, did not result in a significant increase of CHG MIC in S. aureus and E. coli but produced an unstable clinical resistance to tobramycin in E. coli.110 A 4-fold increase was described after 10 days of CHG exposure in E. faecalis.111 These data underline once more the unusual adaptability of gram-negative nosocomial species, also to CHG. Only for S. mutans does it seem to be different. An analysis of 424 clinical isolates of S. mutans from saliva showed that all of them were CHG susceptible with an MIC value ≤ 1 mg/l even though 70% of the subjects had previously used CHG in the oral cavity.112 Similar results were described with MIC values ≤ 1 mg/l from 863 clinical isolates of S. mutans and 53 isolates of S. Page 10

ACCEPTED MANUSCRIPT sobrinus after use of CHG in the oral cavity for up to 7 days.113 A change of susceptibility in S. mutans was also not found after 10 days of CHG exposure.111 In order to describe the average overall exposure of patients of CHG, the different areas of application were analyzed and a possible and realistic exposure estimated. The focus of CHG application is probably the intensive care medicine where most of the indications can be found.

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Hygienic hand wash: Most soaps contain between 2% and 4% CHG. A commonly used product is recommended by the manufacturer to be used with a volume of 5 ml. In clinical practice it seems likely that smaller volumes are used, e.g. 3 ml. Both scenarios will be addressed in Table IV. The total number of indications for hand hygiene in an intensive care unit (ICU) has been described to be 179.114 The assumption in Table IV is that CHG soap is used as recommended in all moments for hand hygiene. For hand hygiene in patient care antimicrobial soaps are not even mentioned in the WHO guideline and can be considered to be dispensable.115

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Hygienic hand disinfection: CHG is often found at 0.5% in propanol-based hand rubs. A hand rub is usually applied with a volume of 3 ml depending on the hand size. The total number of indications for hand hygiene in an intensive care unit (ICU) has been described to be 179 per patient day which includes all shifts and all healthcare workers per patient.114 The assumption in Table IV is that the CHG hand rub is used as recommended in all moments for hand hygiene with a volume of 3 ml. WHO recommends for patient care hand rubs based on alcohol (category IA), additional active ingredients such as CHG are not specifically recommended.115 Body wash: Liquid soaps based on 2% are often used. Patients are usually washed once per day. A volume of 15 ml has been described to be used for washing ICU patients.116 A daily body wash with a soap containing 2% CHG is recommended for patients with CVC to reduce CLABSI.3

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Mouth wash: CHG is usually applied in water at 0.12% (conscious patients) or 0.2% (ventilated patients).117 Manufacturers often recommend a volume of 10 ml to be used in conscious patients. For prevention of ventilator-associated pneumonia a mouth wash may be carried out up to 6 times per day in ventilated patients.118 It is recommended for prevention of ventilatorassociated pneumonia,119 especially for cardiac surgery patients.120

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CVC insertion site care: Application of CHG > 0.5% in alcohol is recommended for skin antisepsis prior to CVC insertion.3 In clinical practice skin antiseptics applied before CVC insertion or during dressing change often contain 2% CHG in 70% iso-propanol. One manufacturer provides an applicator for this specific indication with a volume of 3 ml which is considered sufficient. The frequency of application depends on the type of dressing. When gauze dressings are used it is recommended to change dressing every two day, in transparent dressing it can be up to 7 days.3 Skin antisepsis prior to surgery: 2% CHG is usually applied in combination with 70% isopropanol.7 One manufacturer provides an applicator with a volume of 10.5 ml which is considered sufficient for the majority of applications although larger volumes may be necessary. For the calculation below a volume of 10.5 ml was used. Taking into account the variety of ICUs it was assumed that on average every tenth patient underwent surgery per day requiring skin antisepsis. When in all hand hygiene moments CHG soaps were used, the overall CHG exposure would be between 10.7 and 35.8 g per patient day (Table IV). When a hand rub with 0.5% CHG would be applied for the same hand hygiene moments, the overall exposure would still be 2.7 g per patient day. In this scenario most CHG will be introduced by hand hygiene (not even recommended) whereas the amount used for treatment of CVC puncture sites (category IA) is only 0.03 (CVC Page 11

ACCEPTED MANUSCRIPT insertion) or 0.009 g CHG (puncture site care) per patient day. In the future it may be difficult to explain that a potential lack of efficacy of CHG in CVC puncture site care was evoked by a rather thoughtless and broad use of the agent for all types of applications without a clear benefit. Therefore, it seems only logical to confine the use of CHG very consciously to those applications with a clear benefit and to consider to eliminate it from all applications with a doubtful patient benefit. Perspective for the future

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CHG is a very valuable active agent with solid evidence of effectiveness in preventing specific types of healthcare-associated infections when used as recommended. The most striking example is the benefit of preventing CLABSI when used for treatment of the CVC puncture site. There are however some possibilities to reduce the overall CHG exposure in healthcare without an apparent risk for patient safety. Continued use of CHG for various types of application, including those without good evidence for patient safety, seems to be the larger risk when continuous selection pressure enhances resistance to CHG and possibly also cross-resistance to antibiotics.121 Non-critical or inappropriate use of specific agents such as CHG should therefore be challenged.122 Presented next are some proposals to initiate a “biocidal stewardship” with CHG as an example. The aim is to keep its valuable effect as long as possible for all indications with a definite patient benefit. Proposal 1: For routine hand hygiene eliminate CHG soaps and provide alcohol-based hand rubs and simple soap as recommended by WHO. CHG soaps are not necessary but will be the biggest contributor to the overall exposure when routinely used for hand hygiene.

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Proposal 2: Use alcohol-based hand rubs without CHG. If hand rubs with CHG are routinely used for hand hygiene they will be the second biggest contributor to the overall CHG exposure. WHO does not recommend hand rubs with CHG, and their contribution to the overall efficacy is at least doubtful on dry hands.123

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Proposal 3: For body wash, mouth wash or skin antisepsis prior to surgery the evidence and recommendations are not entirely clear. However, the contribution from these indications to the overall CHG exposure is rather small. It will be an institutional decision based on expected patient benefits whether CHG is still used for these applications or not.

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Proposal 4: Although public data for alcohol-based hand rubs with other types of cationic active ingredients are scarce it seems only logical to additionally review the ingredients for substances such as octenidine, mecetroniumetilsulfate, triclosan or ortho-phenylphenol and evaluate if there is sufficient benefit that outweighs their potential risks (e.g. selection pressure). Proposal 5: A similar evaluation will also be meaningful for other biocidal agents such as triclosan mainly used in antimicrobial soaps, or mupirocin used for nasal decolonization of S. aureus and MRSA. The clinical benefit of triclosan is highly doubtful whereas the clinical benefit of mupirocin is visible but getting smaller. Mupirocin exposure induces resistance,124 high-level mupirocin resistance (mupA carriage) is linked to MDR;125 this could be disseminated among MRSA by plasmids.126 Use of these agents should also be restricted to those applications with an expected patient benefit, and off-label use should be avoided.127 Conclusions Based on the quite high resistance rates in Enterobacter spp., Pseudomonas spp., Proteus spp., Providencia spp. and Enterococcus spp., the ability of Acinetobacter spp., K. pneumoniae and Pseudomonas spp. to adapt to CHG and the potential for cross resistance to some antibiotics it Page 12

ACCEPTED MANUSCRIPT seems perspicacious to restrict the use of CHG to those applications with a clear patient benefit and to eliminate it from applications without any benefit or with a doubtful benefit. Conflict of interest None. References

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114. Steed C, Kelly JW, Blackhurst D, et al. Hospital hand hygiene opportunities: where and when (HOW2)? The HOW2 Benchmark Study. Am J Infect Control 2011;39:19-26. 115. WHO. WHO guidelines on hand hygiene in health care. First Global Patient Safety Challenge Clean Care is Safer Care. Geneva: WHO; 2009. 116. Derde LP, Dautzenberg MJ, Bonten MJ. Chlorhexidine body washing to control antimicrobialresistant bacteria in intensive care units: a systematic review. Intensive Care Med 2012;38:931-939. 117. Bellissimo-Rodrigues WT, Menegueti MG, Gaspar GG, et al. Effectiveness of a dental care intervention in the prevention of lower respiratory tract nosocomial infections among intensive care patients: a randomized clinical trial. Infect Control Hosp Epidemiol 2014;35:1342-1348. 118. Hutchins K, Karras G, Erwin J, Sullivan KL. Ventilator-associated pneumonia and oral care: a successful quality improvement project. Am J Infect Control 2009;37:590-597. 119. Klompas M, Branson R, Eichenwald EC, et al. Strategies to prevent ventilator-associated pneumonia in acute care hospitals: 2014 update. Infect Control Hosp Epidemiol 2014;35 (Suppl 2):S133-S154. 120. Klompas M, Speck K, Howell MD, Greene LR, Berenholtz SM. Reappraisal of routine oral care with chlorhexidine gluconate for patients receiving mechanical ventilation: systematic review and meta-analysis. JAMA Intern Med 2014;174:751-761. 121. Septimus EJ, Schweizer ML. Decolonization in prevention of health care-associated infections. Clin Microbiol Rev 2016;29:201-222. 122. Braoudaki M, Hilton AC. Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents. J Clin Microbiol 2004;42:73-78. 123. Rutter JD, Angiulo K, Macinga DR. Measuring residual activity of topical antimicrobials: is the residual activity of chlorhexidine an artefact of laboratory methods? J Hosp Infect 2014;88:113-115. 124. Hetem DJ, Vogely HC, Severs TT, Troelstra A, Kusters JG, Bonten MJ. Acquisition of high-level mupirocin resistance in CoNS following nasal decolonization with mupirocin. J Antimicrob Chemother 2015;70:1182-1184. 125. Poovelikunnel T, Gethin G, Humphreys H. Mupirocin resistance: clinical implications and potential alternatives for the eradication of MRSA. J Antimicrob Chemother 2015;70:2681-2692. 126. Perez-Roth E, Potel-Alvarellos C, Espartero X, Constela-Carames L, Mendez-Alvarez S, AlvarezFernandez M. Molecular epidemiology of plasmid-mediated high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus in four Spanish health care settings. Int J Med Microbiol 2013;303:201-204. 127. Lepainteur M, Royer G, Bourrel AS, et al. Prevalence of resistance to antiseptics and mupirocin among invasive coagulase-negative staphylococci from very preterm neonates in NICU: the creeping threat? J Hosp Infect 2013;83:333-336. 128. Koljalg S, Naaber P, Mikelsaar M. Antibiotic resistance as an indicator of bacterial chlorhexidine susceptibility. J Hosp Infect 2002;51:106-113. 129. Naparstek L, Carmeli Y, Chmelnitsky I, Banin E, Navon-Venezia S. Reduced susceptibility to chlorhexidine among extremely-drug-resistant strains of Klebsiella pneumoniae. J Hosp Infect 2012;81:15-19. 130. Kurihara T, Sugita M, Motai S, Kurashige S. In vitro induction of chlorhexidine- and benzalkonium-resistance in clinically isolated Pseudomonas aeruginosa. Kansenshogaku Zasshi 1993;67:202-206. 131. Russell AD, Mills AP. Comparative sensitivity and resistance of some strains of Pseudomonas aeruginosa and Pseudomonas stutzeri to antibacterial agents. J Clin Pathol 1974;27:463-466. 132. Lambert RJ, Joynson J, Forbes B. The relationship and susceptibilities of some industrial laboratory and clinical isolates of Pseudomonas aeruginosa to some antibiotics and biocides. J Appl Microbiol 2001;91:972-984. 133. Kawamura-Sato K, Wachino J, Kondo T, Ito H, Arakawa Y. Correlation between reduced susceptibility to disinfectants and multidrug resistance among clinical isolates of Acinetobacter species. J Antimicrob Chemother 2010;65:1975-1983. Page 19

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134. Chung YK, Kim JS, Lee SS, et al. Effect of daily chlorhexidine bathing on acquisition of carbapenem-resistant Acinetobacter baumannii (CRAB) in the medical intensive care unit with CRAB endemicity. Am J Infect Control 2015;43:1171-1177. 135. Higgins CS, Murtough SM, Williamson E, et al. Resistance to antibiotics and biocides among non-fermenting Gram-negative bacteria. Clin Microbiol Infect 2001;7:308-315. 136. Okuda T, Endo N, Osada Y, Zen-Yoji H. Outbreak of nosocomial urinary tract infections caused by Serratia marcescens. J Clin Microbiol 1984;20:691-695. 137. Stickler DJ. Chlorhexidine resistance in Proteus mirabilis. J Clin Pathol 1974;27:284-287. 138. Ho CM, Li CY, Ho MW, Lin CY, Liu SH, Lu JJ. High rate of qacA- and qacB-positive methicillinresistant Staphylococcus aureus isolates from chlorhexidine-impregnated catheter-related bloodstream infections. Antimicrob Agents Chemother 2012;56:5693-5697. 139. Ye JZ, Yu X, Li XS, et al. Antimicrobial resistance characteristics of and disinfectant-resistant gene distribution in Staphylococcus aureus isolates from male urogenital tract infection. Zhonghua nan ke xue 2014;20:630-636. 140. Munoz-Gallego I, Infiesta L, Viedma E, Perez-Montarelo D, Chaves F. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Glob Antimicrob Resist 2016;4:65-69. 141. Sheng WH, Wang JT, Lauderdale TL, Weng CM, Chen D, Chang SC. Epidemiology and susceptibilities of methicillin-resistant Staphylococcus aureus in Taiwan: emphasis on chlorhexidine susceptibility. Diagn Microbiol Infect Dis 2009;63:309-313. 142. McDanel JS, Murphy CR, Diekema DJ, et al. Chlorhexidine and mupirocin susceptibilities of methicillin-resistant Staphylococcus aureus from colonized nursing home residents. Antimicrob Agents Chemother 2013;57:552-558. 143. Hijazi K, Mukhopadhya I, Abbott F, et al. Susceptibility to chlorhexidine amongst multidrugresistant clinical isolates of Staphylococcus epidermidis from bloodstream infections. Int J Antimicrob Agents 2016;48:86-90. 144. Valenzuela AS, Benomar N, Abriouel H, Canamero MM, Lopez RL, Galvez A. Biocide and copper tolerance in enterococci from different sources. J Food Prot 2013;76:1806-1809. 145. Suller MTE, Russell AD. Antibiotic and biocide resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcus. J Hosp Infect 1999;43:281-291. 146. Shamsudin MN, Alreshidi MA, Hamat RA, Alshrari AS, Atshan SS, Neela V. High prevalence of qacA/B carriage among clinical isolates of meticillin-resistant Staphylococcus aureus in Malaysia. J Hosp Infect 2012;81:206-208. 147. Miyazaki NH, Abreu AO, Marin VA, Rezende CA, Moraes MT, Villas Boas MH. The presence of qacA/B gene in Brazilian methicillin-resistant Staphylococcus aureus. Memorias do Instituto Oswaldo Cruz 2007;102:539-540. 148. Ho J, Branley J. Prevalence of antiseptic resistance genes qacA/B and specific sequence types of methicillin-resistant Staphylococcus aureus in the era of hand hygiene. J Antimicrob Chemother 2012;67:1549-1550. 149. Ghasemzadeh-Moghaddam H, van Belkum A, Hamat RA, van Wamel W, Neela V. Methicillinsusceptible and -resistant Staphylococcus aureus with high-level antiseptic and low-level mupirocin resistance in Malaysia. Microb Drug Resist 2014;20:472-477. 150. Wang C, Cai P, Zhan Q, Mi Z, Huang Z, Chen G. Distribution of antiseptic-resistance genes qacA/B in clinical isolates of meticillin-resistant Staphylococcus aureus in China. J Hosp Infect 2008;69:393-394. 151. Cho OH, Baek EH, Bak MH, et al. The effect of targeted decolonization on methicillin-resistant Staphylococcus aureus colonization or infection in a surgical intensive care unit. Am J Infect Control 2016;44:533-538. 152. Zheng R, Wang M, He B, et al. Identification of active efflux system gene qacA/B in methicillin-resistant Staphylococcus aureus and its significance. Zhong nan da xue xue bao Yi xue ban 2009;34:537-542. Page 20

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153. Li T, Song Y, Zhu Y, Du X, Li M. Current status of Staphylococcus aureus infection in a central teaching hospital in Shanghai, China. BMC Microbiol 2013;13:153. 154. Aykan SB, Caglar K, Engin ED, Sipahi AB, Sultan N, Yalinay Cirak M. Investigation of the presence of disinfectant resistance genes qaca/b in nosocomial methicillin-resistant Staphylococcus aureus isolates and evaluation of their in vitro disinfectant susceptibilities. Mikrobiyoloji bulteni 2013;47:1-10. 155. Lu Z, Chen Y, Chen W, et al. Characteristics of qacA/B-positive Staphylococcus aureus isolated from patients and a hospital environment in China. J Antimicrob Chemother 2015;70:653-657. 156. Rondeau C, Chevet G, Blanc DS, et al. Current molecular epidemiology of methicillin-resistant Staphylococcus aureus in elderly french people: troublesome clones on the horizon. Front Microbiol 2016;7:31. 157. Warren DK, Prager M, Munigala S, et al. Prevalence of qacA/B genes and mupirocin resistance among methicillin-resistant Staphylococcus aureus (MRSA) isolates in the setting of chlorhexidine bathing without mupirocin. Infect Control Hosp Epidemiol 2016;37:590-597. 158. Schlett CD, Millar EV, Crawford KB, et al. Prevalence of chlorhexidine-resistant methicillinresistant Staphylococcus aureus following prolonged exposure. Antimicrob Agents Chemother 2014;58:4404-4410. 159. Mc Gann P, Milillo M, Kwak YI, Quintero R, Waterman PE, Lesho E. Rapid and simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and mupA in clinical isolates of methicillin-resistant Staphylococcus aureus. Diagn Microbiol Infect Dis 2013;77:270-272. 160. Shigeta S, Yasunaga Y, Honzumi K, Okamura H, Kumata R, Endo S. Cerebral ventriculitis associated with Achromobacter xylosoxidans. J Clin Pathol 1978;31:156-161. 161. McAllister TA, Lucas CE, Mocan H, et al. Serratia marcescens outbreak in a paediatric oncology unit traced to contaminated chlorhexidine. Scot Med J 1989;34:525-528. 162. Dance DAB, Pearson AD, Seal DV, Lowes JA. A hospital outbreak caused by a chlorhexidine and antibiotic-resistant Proteus mirabilis. J Hosp Infect 1987;10:10-16. 163. Batra R, Cooper BS, Whiteley C, Patel AK, Wyncoll D, Edgeworth JD. Efficacy and limitation of a chlorhexidine-based decolonization strategy in preventing transmission of methicillin-resistant Staphylococcus aureus in an intensive care unit. Clin Infect Dis 2010;50:210-217. 164. Prag G, Falk-Brynhildsen K, Jacobsson S, Hellmark B, Unemo M, Soderquist B. Decreased susceptibility to chlorhexidine and prevalence of disinfectant resistance genes among clinical isolates of Staphylococcus epidermidis. APMIS 2014;122:961-967. 165. Ben Saida N, Marzouk M, Ferjeni A, Boukadida J. A three-year surveillance of nosocomial infections by methicillin-resistant Staphylococcus haemolyticus in newborns reveals the disinfectant as a possible reservoir. Pathologie-biologie 2009;57:e29-35.

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Table I: Range of CHG MIC-values and medians obtained from clinical samples for various nosocomial pathogens; for S. aureus a MIC value of 1 mg/l was proposed in addition in 2015 101. Country

Number of isolates or strains

Range of MICs

Median MIC

E. coli

UK33

120 clinical isolates

1 – 5 mg/l*

n.a.

UK33

10 clinical strains

0.5 – 5 mg/l*

2.5 mg/l*

UK33

5 laboratory strains

1.5 – 2 mg/l*

Estonia128

10 clinical isolates

2- 16 mg/l

2 mg/l

Klebsiella spp.

UK33

8 clinical strains

25 - 75 mg/l*

45 mg/l*

K. pneumoniae

Mexico34

35 ESBL clinical isolates

16 – 64 mg/l

32 mg/l

UK27

5 clinical isolates

10 – 200 mg/l

30 mg/l

Israel129

126 XDR clinical isolates

8 - > 256 mg/l

140 mg/l**

Estonia128

10 clinical isolates

16 – 32 mg/l

16 mg/l

UK32

64 clinical isolates

4 – 128 mg/l

32 mg/l

Enterobacter spp.

UK33

8 clinical strains

10 – 75 mg/l*

48 mg/l*

16 mg/l (Enterobacter spp.)21

Salmonella spp.

China35

195 isolates from chicken and egg production

< 4 – 64 mg/l

16 mg/l

32 mg/l21

Pseudomonas spp.

UK33

95 clinical isolates

≤ 25 – 100 mg/l

25 mg/l

P. aeruginosa

UK26

35 clinical isolates

10 – 800 mg/l

200 mg/l

50 mg/l (P. aeruginosa)22

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Epidemiological cut-off value for CHG resistance (MIC)

SC

64 mg/l21

1.5 mg/l*

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Bacterial genus / species

64 mg/l (K. pneumoniae)21

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178 clinical isolates

78 – 625 mg/l

312 mg/l

Japan22

317 clinical isolates

3 – 400 mg/l

100 mg/l

UK131

16 clinical strains

25 – 50 mg/l*

n.a.

UK132

20 clinical strains

< 2 – 39 mg/l

19 mg/l

P. stutzeri

UK131

11 clinical strains

< 10 – 50 mg/l*

< 10 mg/l*

Acinetobacter spp.

Japan133

283 clinical strains

10 – 400 mg/l

10 mg/l

A. baumannii

South Korea134

98 carbapenem-resistant clinical isolates

8 – 64 mg/l

UK135

2 clinical strains

125 – 175 mg/l

150 mg/l

A. anitratus

UK33

1 reference strain

20 mg/l*

n.a.

S. marcescens

UK33

2 reference strains

2.5 – 25 mg/l

14 mg/l

Japan136

131 clinical strains

< 3.12 – 400 mg/l

3.12 mg/l (serotype O12/14)

P. mirabilis

48 clinical isolates

UK26

SC

n.a.

32 mg/l

M AN U

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EP

UK33

n.a.

100 mg/l (serotype O2/3) 12.5 mg/l (serotype O13) 6.25 mg/l (serotype NT)

25 – 75 mg/l*

50 mg/l*

181 clinical isolates

10 – 1600 mg/l

200 mg/l

UK27

14 clinical isolates

10 – 1600 mg/l

800 mg/l

UK137

104 clinical isolates

10 – 800 mg/l

50 mg/l

UK33

1 reference strain

125 mg/l*

n.a.

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Proteus spp.

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Japan130

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n.a.

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156 clinical isolates

0.125 – 4 mg/l

1 mg/l

Estonia128

10 clinical isolates

0.5 – 1 mg/l

1 mg/l

China139

152 clinical isolates

0.25 – 16 mg/l

2 mg/l

Hong Kong64

82 isolates from nurses

1 – 8 mg/l

2 mg/l

Spain140

134 clinical isolates

< 0.125 – 4 mg/l

1 mg/l

Taiwan59

240 clinical isolates

0.5 – 16 mg/l

n.a.

Taiwan141

206 clinical isolates

0.125 – 16 mg/l

USA142

829 clinical isolates

0.5 – 4 mg/l

2 mg/l

Coagulasenegative Staphylococcus spp. (CNS)

China64

146 isolates from nurses and the general population

0.5 – 32 mg/l

2 mg/l

France127

51 clinical isolates

0.5 – 8 mg/l

2 mg/l

S. epidermidis

UK143

25 clinical isolates

E. faecium

Spain144

VRE

SC

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4 mg/l

165 isolates from the environment and patients

2.5 – 2500 mg/l

n.a.

Estonia128

10 clinical isolates

2 – 8 mg/l

4 mg/l

Spain144

107 isolates from the environment and patients

2.5 – 2500 mg/l

n.a.

UK145

5 clinical isolates

4 – 6 mg/l

5 mg/l

EP

E. faecalis

2 mg/l

2 – 4 mg/l

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MRSA

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8 mg/l21

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Taiwan138

S. aureus

n.a.

32 mg/l (E. faecium)21 64 mg/l (E. faecalis)21

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MRSA-isolates (n) qacA/B detection rate Reference

Malaysia

60

83.3%

China

53

83.0%

Brazil

74

80%

Australia

151

78.6%

Malaysia

95

Korea

465

Turkey

28

Europa

297

China

M AN U

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Country

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Table II: Detection rates of qacA/B in MRSA isolates.

146 70

147 148 149

65%

80

64.3%

60

62.2%

74

131

61.1%

150

96

43.8%

138

894

41.6%

81

Republic of Korea 169

37.7%

151

Japan

283

33.9%

82

Japan

334

32.6%

75

Serbia

50

32%

66

Scotland

38

26.3%

65

Taiwan

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Asia

EP

70.5%

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240

23.8%

59

China

80

23.8%

152

USA

66

18%

76

China

414

15.7%

Turkey

69

11.6%

South Korea

456

8.8%

Scotland

120

8.3%

China

321

France

39

USA

504

Spain

134

Canada USA

SC

M AN U

153 154 80 84

7.8%

155

7.7%

156

7.1%

157

2.2%

140

334

2.1%

83

341

1.5%

158

1968

0.9%

159

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USA

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Taiwan

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Table III: Infections associated with resistance to CHG or the presence of the qacA/B gene.

Serratia marcescens

Twelve cases of bacteremia in ten patients; three patients died.

Patients on a paediatric oncology unit with Hickman-lines

Proteus mirabilis

88 cases of urinary tract infections and two other types of infection.

Patients on general medical and geriatric wards; 75% of the urinary tract infections were catheter-associated

Methicillinresistant S. aureus

One case of recurrent cutaneous abscess

Patient with a first cutaneous infection on the left knee followed by a similar infection nine weeks later on the left foot

Methicillinresistant S. aureus

517 patients admitted with an MRSA infection, 347 patients acquired an MRSA infection

Source of infection and role of CHG resistance Reference 160 Eleven strains of A. xylosoxidans were identified from cerebrospinal fluid. Off them, seven strains were able to survive in a solution of 2% CHG, three strains in 1% CHG, and one strain in 0.1% CHG. The species was isolated from a container for disposal of surgical instruments which contained a 0.1% CHG solution. CHG at 0.1% was in addition used for surgical scrubbing and for the preoperative treatment of skin. The source of the outbreak was a container close to the 161 patients filled with 0.5% CHG in water. The container was used to store clamps which were used during disconnection to avoid air uptake. The solution was renewed daily. The container, however, was neither cleaned or processed 162 The isolate was multidrug- and chlorhexidine resistant. Resistance to CHG was assumed when an isolates was able to survive in 200 mg/l CHG. The outbreak was suspected to be caused by the widespread use of CHG for various types of antiseptic treatment including hand hygiene. 20 The patient was in a study group that received 4% chlorhexidine soap for weekly showering. He had used chlorhexidine once or twice before his first episode and four or five times prior to the second episode. The first clinical isolate (PFGE type USA 300) was negative for the chlorhexidine resistance genes (qacA/B), but the second one was positive (also PFGE type USA 300). MRSA carrier were treated with an antiseptic protocol: 163 1% CHG applied to nostrils, around the mouth, and at tracheostomy sites 4 times daily; 1% CHA applied daily to groin, axillae, and skinfolds; 4% CHG for daily

RI PT

Patient population Patients on a neurosurgical unit after craniotomy or trepanation

SC

Type and number of infections Eleven cases of bacterial ventriculitis

AC C

EP

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M AN U

Bacterial species Achromobacter xylosoxidans

Two intensive care units

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SC

M AN U

Patients with prosthetic joint infection (61) or surgical site infections after cardiac surgery (31) Patients on a neonatal intensive care unit

EP

Methicillinresistant S. haemolyticus

92 cases of joint or wound infections; 27 additional isolates came from the skin of the chest prior to cardiac surgery 42 clinical isolates; 15 from blood cultures, 14 from vascular catheters, 11 from tracheal tubes and 2 from cerebrospinal fluid. Eight neonates died from the infection.

AC C

Staphylococcus epidermidis

washing. The outbreak strain carried qacA/B genes and demonstrated 3-fold increased chlorhexidine MBCs in vitro. The antiseptic protocol reduced acquisition of non-outbreak MRSA strains by 70% but significantly increased transmission of the outbreak MRSA strain. Depending on the type of wound infection the rate of resistance to CHG varied between 7% and 68%. Resistance to CHG was often associated with the presence of the qacA/B gene Two isolates were detected in open bottles of a multiple use disinfectant based on 1% CHG and 0.2% QAC. The commercial product was used for hand washing. Both isolates were qacA/B carrier and considered to be CHG resistant. Even in four new unopened bottles of the same product different gramnegative species and S. hominis were identified.

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Table IV: Estimated overall CHG exposure of patients on ICU; *in alcohol (e.g. in iso-propanol). Frequency of Typical concentration use of CHG

Estimated amount of product used

Used amount of CHG per application

Hand wash Hand wash Hand disinfection Body wash Mouth wash in ventilated patients Mouth wash in conscious patients CVC insertion site care

4% 2% 0.5%*

179 per day 179 per day 179 per day

5 ml 3 ml 3 ml

35.8 g 10.74 g 0.015 g

Overall CHG load per patient per day 35.8 g 10.74 g 2.685 g

2% 0.2%

1 per day 6 per day

15 ml 10 ml

0.3 g 0.12 g

0.3 g 0.12 g

0.12%

3 per day

10 ml

0.036 g

0.036 g

2%*

Once every 2 days (gauze dressing) 1 per 10 days

3 ml

0.06 g

0.03 g

Once every 7 days (transparent dressing)

3 ml

2%*

SC

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10.5 ml

0.21 g

EP

2%*

0.06 g

0.021 g

0.009 g

AC C

Skin antisepsis before surgery CVC insertion site care

RI PT

CHG used for

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