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.

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

Günter Kampf 1,2*

1Knieler
und Team GmbH, Infection Control Science, Kattrepelsbrücke 1, 20095 Hamburg,
Germany; email: g.kampf@knielerundteam.de; tel. +49 40 328907275

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2Ernst-Moritz-Arndt Universität, Institut für Hygiene und Umweltmedizin, Walter-Rathenau-
Straße 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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Summary
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 multi-
resistant isolates such as XDR K. pneumoniae. Isolates with a higher MIC are often less

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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

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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

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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

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and to eliminate it from applications without any benefit or with a doubtful benefit.
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Introduction
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

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catheter-associated bloodstream infections.3,4
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

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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

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respected study on the effect of compliance in hand hygiene on the incidence of healthcare-
associated infections. 9
The overall risk for an acquired resistance to biocidal agents such as CHG is considered to be

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small as long as the antiseptics are used correctly.10 At the same time a number of outbreaks has
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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
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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
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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
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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.
Epidemiological cut-off values to determine resistance to CHG
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The greater use of CHG warrants surveillance for resistance.20 In order to identify biocide
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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

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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
Susceptibility to CHG in relation to epidemiological cut-off values

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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

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evidence for resistance in clinical applications.24
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

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(≤ 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
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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
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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
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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
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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
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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
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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.
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

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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.

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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

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Pseudomonas spp. was also detected as a contaminant in CHG solutions of unknown strength in
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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
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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
Pseudomonas spp. can adapt or develop resistance to CHG quite easily.37 After repetitive
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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
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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.
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aeruginosa when present in biofilm.43

Providencia spp.
Resistance to CHG in Providencia spp. was described frequently especially in isolates from
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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%
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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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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.
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.

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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

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(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.
An ESBL S. marcescens was able to persist and continuously contaminate a CHG stock solution of

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an unknown concentration in a Mexican children’s hospital and may have contributed to an
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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
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with an unknown function.53

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
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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
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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
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Staphylococcus aureus
An overview of MIC values obtained in clinical isolates of S. aureus is provided in Table I.
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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

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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
Coagulase-negative Staphylococcus spp. (CNS)

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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

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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

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mainly found in clinical material. To what extent a higher MIC value in Enterococcus spp. changes
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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
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Common resistance genes

Multidrug efflux pumps can be divided into 5 protein families depending on their energy
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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
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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
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S. aureus isolates with 38 of them being HA-MRSA, 25 CA-MRSA, 6 VISA and 25 MSSA.65
qacA/B gene
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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
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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
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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.
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

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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

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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
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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
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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
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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
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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
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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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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
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

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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
Other studies indicate that cross resistance between CHG and antibiotics does occur. An analysis

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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

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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

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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
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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
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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
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Japan an outbreak of 7 cases of catheter-associated urinary tract infection caused by multi-

resistant P. aeruginosa was analyzed. The outbreak strain was resistant to CHG and at the same
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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
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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
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Cross resistance has also been described in Gram-positive species. When healthcare workers
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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
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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

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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

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pressure has optimized the content of certain plasmids despite their large size and complex
organization.103
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

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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
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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,
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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.
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CHG exposure in healthcare

In the UK the MIC values of 251 clinical isolates to CHG were opposed to the magnitude of CHG
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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
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When different bacterial species (S. aureus, E. coli, P. aeruginosa, S. marcescens, C. albicans) are
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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.

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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.
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

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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

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mentioned in the WHO guideline and can be considered to be dispensable.115
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

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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

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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
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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.
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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 ventilator-
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associated pneumonia,119 especially for cardiac surgery patients.120

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
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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
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days.3
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Skin antisepsis prior to surgery: 2% CHG is usually applied in combination with 70% iso-
propanol.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
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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
CHG is a very valuable active agent with solid evidence of effectiveness in preventing specific

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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

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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

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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.

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Proposal 1: For routine hand hygiene eliminate CHG soaps and provide alcohol-based hand rubs
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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.
Proposal 2: Use alcohol-based hand rubs without CHG. If hand rubs with CHG are routinely used
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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
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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).
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Proposal 5: A similar evaluation will also be meaningful for other biocidal agents such as
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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
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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.
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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.

Bacterial genus / Country Number of isolates or Range of MICs Median MIC Epidemiological cut-off

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species strains value for CHG resistance
(MIC)

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E. coli UK33 120 clinical isolates 1 – 5 mg/l* n.a. 64 mg/l21

UK33 10 clinical strains 0.5 – 5 mg/l* 2.5 mg/l*

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UK33 5 laboratory strains 1.5 – 2 mg/l* 1.5 mg/l*

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Estonia128 10 clinical isolates 2- 16 mg/l 2 mg/l

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Klebsiella spp. UK33 8 clinical strains 25 - 75 mg/l* 45 mg/l* 64 mg/l (K.
pneumoniae)21
K. pneumoniae Mexico34 35 ESBL clinical isolates 16 – 64 mg/l 32 mg/l

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UK27 5 clinical isolates 10 – 200 mg/l 30 mg/l

Israel129 126 XDR clinical isolates 8 - > 256 mg/l 140 mg/l**

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Estonia128 10 clinical isolates 16 – 32 mg/l 16 mg/l

UK32 64 clinical isolates 4 – 128 mg/l 32 mg/l

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Enterobacter spp. UK33 8 clinical strains 10 – 75 mg/l* 48 mg/l* 16 mg/l (Enterobacter
spp.)21
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Salmonella spp. China35 195 isolates from chicken < 4 – 64 mg/l 16 mg/l 32 mg/l21
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and egg production

Pseudomonas spp. UK33 95 clinical isolates ≤ 25 – 100 mg/l 25 mg/l 50 mg/l (P.
aeruginosa)22
P. aeruginosa UK26 35 clinical isolates 10 – 800 mg/l 200 mg/l

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Japan130 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.

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UK132 20 clinical strains < 2 – 39 mg/l 19 mg/l

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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 n.a.

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A. baumannii South 98 carbapenem-resistant 8 – 64 mg/l 32 mg/l
Korea134 clinical isolates

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UK135 2 clinical strains 125 – 175 mg/l 150 mg/l

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A. anitratus UK33 1 reference strain 20 mg/l* n.a.

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S. marcescens UK33 2 reference strains 2.5 – 25 mg/l 14 mg/l n.a.

Japan136 131 clinical strains < 3.12 – 400 mg/l 3.12 mg/l (serotype O12/14)

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100 mg/l (serotype O2/3)

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6.25 mg/l (serotype NT)

Proteus spp. UK33 48 clinical isolates 25 – 75 mg/l* 50 mg/l* n.a.

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UK26 181 clinical isolates 10 – 1600 mg/l 200 mg/l

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UK27 14 clinical isolates 10 – 1600 mg/l 800 mg/l

P. mirabilis UK137 104 clinical isolates 10 – 800 mg/l 50 mg/l

UK33 1 reference strain 125 mg/l* n.a.

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S. aureus Taiwan138 156 clinical isolates 0.125 – 4 mg/l 1 mg/l 8 mg/l21

Estonia128 10 clinical isolates 0.5 – 1 mg/l 1 mg/l

China139 152 clinical isolates 0.25 – 16 mg/l 2 mg/l

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Hong Kong64 82 isolates from nurses 1 – 8 mg/l 2 mg/l

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MRSA Spain140 134 clinical isolates < 0.125 – 4 mg/l 1 mg/l

Taiwan59 240 clinical isolates 0.5 – 16 mg/l n.a.

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Taiwan141 206 clinical isolates 0.125 – 16 mg/l 2 mg/l

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USA142 829 clinical isolates 0.5 – 4 mg/l 2 mg/l

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Coagulase- China64 146 isolates from nurses 0.5 – 32 mg/l 2 mg/l n.a.
negative and the general population
Staphylococcus

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spp. (CNS) France127 51 clinical isolates 0.5 – 8 mg/l 2 mg/l

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S. epidermidis UK143 25 clinical isolates 2 – 4 mg/l 4 mg/l

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E. faecium Spain144 165 isolates from the 2.5 – 2500 mg/l n.a. 32 mg/l (E. faecium)21
environment and patients
64 mg/l (E. faecalis)21
E. faecalis Estonia128 10 clinical isolates 2 – 8 mg/l 4 mg/l
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Spain144 107 isolates from the 2.5 – 2500 mg/l n.a.
environment and patients
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VRE UK145 5 clinical isolates 4 – 6 mg/l 5 mg/l

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

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

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Malaysia 60 83.3% 146

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China 53 83.0% 70

Brazil 74 80% 147

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Australia 151 78.6% 148

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149
Malaysia 95 70.5%

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Korea 465 65% 80

Turkey 28 64.3% 60

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Europa 297 62.2% 74

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China 131 61.1% 150

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Taiwan 96 43.8% 138

Asia 894 41.6% 81

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Republic of Korea 169 37.7% 151

Japan 283 33.9% 82

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Japan 334 32.6% 75

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Serbia 50 32% 66

Scotland 38 26.3% 65

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Taiwan 240 23.8% 59

China 80 23.8% 152

USA 66 18% 76

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China 414 15.7% 153

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Turkey 69 11.6% 154

South Korea 456 8.8% 80

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Scotland 120 8.3% 84

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China 321 7.8% 155

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France 39 7.7% 156

USA 504 7.1% 157

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Spain 134 2.2% 140

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Canada 334 2.1% 83

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USA 341 1.5% 158

USA 1968 0.9% 159

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

Bacterial species Type and number of infections Patient population Source of infection and role of CHG resistance Reference
Achromobacter Eleven cases of bacterial Patients on a Eleven strains of A. xylosoxidans were identified from 160

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xylosoxidans ventriculitis neurosurgical unit after cerebrospinal fluid. Off them, seven strains were able
craniotomy or trepanation to survive in a solution of 2% CHG, three strains in 1%
CHG, and one strain in 0.1% CHG. The species was

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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

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scrubbing and for the preoperative treatment of skin.
Serratia Twelve cases of bacteremia in Patients on a paediatric The source of the outbreak was a container close to the 161
marcescens ten patients; three patients died. oncology unit with patients filled with 0.5% CHG in water. The container

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Hickman-lines was used to store clamps which were used during

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disconnection to avoid air uptake. The solution was
renewed daily. The container, however, was neither
cleaned or processed

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Proteus mirabilis 88 cases of urinary tract Patients on general The isolate was multidrug- and chlorhexidine 162

infections and two other types of medical and geriatric resistant. Resistance to CHG was assumed when an
infection. wards; 75% of the urinary isolates was able to survive in 200 mg/l CHG. The

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tract infections were outbreak was suspected to be caused by the

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catheter-associated widespread use of CHG for various types of antiseptic
treatment including hand hygiene.
Methicillin- One case of recurrent cutaneous Patient with a first The patient was in a study group that received 4% 20
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resistant S. abscess cutaneous infection on the chlorhexidine soap for weekly showering. He had used
aureus left knee followed by a chlorhexidine once or twice before his first episode
similar infection nine and four or five times prior to the second episode. The
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weeks later on the left foot first clinical isolate (PFGE type USA 300) was negative
for the chlorhexidine resistance genes (qacA/B), but
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the second one was positive (also PFGE type USA 300).
Methicillin- 517 patients admitted with an Two intensive care units MRSA carrier were treated with an antiseptic protocol: 163
resistant S. MRSA infection, 347 patients 1% CHG applied to nostrils, around the mouth, and at
aureus acquired an MRSA infection tracheostomy sites 4 times daily; 1% CHA applied
daily to groin, axillae, and skinfolds; 4% CHG for daily
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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

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significantly increased transmission of the outbreak
MRSA strain.
Staphylococcus 92 cases of joint or wound Patients with prosthetic Depending on the type of wound infection the rate of 164

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epidermidis infections; 27 additional isolates joint infection (61) or resistance to CHG varied between 7% and 68%.
came from the skin of the chest surgical site infections Resistance to CHG was often associated with the

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prior to cardiac surgery after cardiac surgery (31) presence of the qacA/B gene
Methicillin- 42 clinical isolates; 15 from Patients on a neonatal Two isolates were detected in open bottles of a 165

resistant S. blood cultures, 14 from vascular intensive care unit multiple use disinfectant based on 1% CHG and 0.2%

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haemolyticus catheters, 11 from tracheal tubes QAC. The commercial product was used for hand
and 2 from cerebrospinal fluid. washing. Both isolates were qacA/B carrier and

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Eight neonates died from the considered to be CHG resistant. Even in four new
infection. unopened bottles of the same product different gram-
negative 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).

CHG used for Typical Frequency of Estimated Used amount Overall CHG
concentration use amount of of CHG per load per

PT
of CHG product used application patient per
day
Hand wash 4% 179 per day 5 ml 35.8 g 35.8 g

RI
Hand wash 2% 179 per day 3 ml 10.74 g 10.74 g
Hand 0.5%* 179 per day 3 ml 0.015 g 2.685 g
disinfection

SC
Body wash 2% 1 per day 15 ml 0.3 g 0.3 g
Mouth wash 0.2% 6 per day 10 ml 0.12 g 0.12 g

U
in ventilated
patients

AN
Mouth wash 0.12% 3 per day 10 ml 0.036 g 0.036 g
in conscious
patients

M
CVC insertion 2%* Once every 2 3 ml 0.06 g 0.03 g
site care days (gauze

D
dressing)
Skin 2%* 1 per 10 days 10.5 ml 0.21 g 0.021 g

TE
antisepsis
before
surgery
EP
CVC insertion 2%* Once every 7 3 ml 0.06 g 0.009 g
site care days
(transparent
C

dressing)
AC

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