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. 2019 Mar 7;6(7):307–320. doi: 10.15698/mic2019.07.683

Adaptive bacterial response to low level chlorhexidine exposure and its implications for hand hygiene

Günter Kampf 1,*
PMCID: PMC6600115  PMID: 31294043

Abstract

Chlorhexidine digluconate (CHG) is commonly used in healthcare, e.g. in skin antiseptics, antimicrobial soaps, alcohol-based hand rubs and oral or wound antiseptics. Aim of the literature review was to evaluate the potential of bacteria to adapt to low level CHG exposure. A maximum 4fold MIC increase to CHG was found after low level exposure in most of the 71 evaluated bacterial species. A strong adaptive mostly stable MIC change was described in strains or isolates of the healthcare-associated species E. coli, S. marcescens and P. aeruginosa (up to 500fold, 128fold or 32fold, respectively). The highest MIC values after adaptation were 2,048 mg/l (S. marcescens) and 1,024 mg/l (P. aeruginosa). A new resistance to tetracycline, gentamicin, meropeneme or triclosan was found in some adapted isolates. In E. coli horizontal gene transfer was induced (sulfonamide resistance by conjugation), pointing out an additional risk of sublethal CHG. The use of CHG in patient care - but also all other settings such as consumer products and households - should therefore be critically assessed and restricted to indications with a proven health benefit or justifiable public health benefits. Additional CHG has no health benefit when used in alcohol-based hand rubs and is not recommended by the WHO. For routine hand washing of soiled hands the use of plain soap is sufficient, CHG in soaps has no health benefit. In surgical hand antisepsis alcohol-based hand rubs should be preferred to CHG soaps. Implementation of these principles will help to reduce avoidable selection pressure.

Keywords: chlorhexidine digluconate, adaptation, resistance, cross-tolerance, low level exposure, MIC values

INTRODUCTION

Chlorhexidine digluconate (CHG) is a commonly used antiseptic agent in human healthcare and veterinary medicine, mainly used for hand hygiene (e.g. at 2% - 4% as the only active agent in antiseptic soaps or at 0.5% or 1% as an additional active agent in alcohol-based hand rubs), in alcohol-based skin antiseptics at 2% and in mouth rinse solutions at 0.12% - 0.2% [1]. The widespread CHG use in various types of applications has probably lead to an increase of acquired bacterial resistances, mainly in Gram-negative species such as Pseudomonas aeruginosa (minimal inhibitory concentration (MIC) of up to 800 mg/l), Serratia marcescens (MIC of up to 400 mg/l) or Klebsiella pneumoniae (MIC of up to 256 mg/l) [1]. In some types of applications such as skin antiseptics CHG has been shown to reduce healthcare associated infections, e.g. catheter-associated bloodstream infections [2]. Recent evidence also suggests a contribution to the prevention of surgical site infections [3] although the single effect of CHG for this application is still under controversial debate [4–6].

Its widespread use in hand hygiene by healthcare workers in many countries suggests to look specifically at all possible applications in this area. The WHO has published a recommendation on hand hygiene for healthcare in 2009 with the aim to reduce healthcare-associated infections [7]. Three types of applications can be distinguished. The use of alcohol-based hand rubs is recommended on clean hands in five specific clinical situations: before touching a patient, before clean or aseptic procedures, after body fluid exposure, after touching a patient and after touching patient surroundings [7, 8]. Hand washing with either plain soap or antiseptic soap and water is recommended for visibly soiled hands or in case of contamination with spore-forming bacteria such as Clostridium difficile [7]. The third type of application is in the surgical theater. Healthcare workers should decontaminate their hands prior to donning sterile surgical gloves with either alcohol-based hand rubs (surgical hand disinfection) or with antimicrobial soaps (surgical scrubbing) [7].

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In the past years there is an increasing concern on the development of resistance not only to antibiotics but also to antiseptic agents which are essential to limit the spread of multidrug-resistant pathogens in healthcare [9, 10]. Some antiseptic agents are more likely than others to cause a bacterial tolerance or even resistance [11]. Aim of the review is therefore to evaluate the potential of CHG to cause an adaptive bacterial response during exposure to sublethal concentrations and to propose reasonable implications for the use of CHG in hand hygiene.

RESULTS

General remark

The magnitude of any adaptive response to CHG is expressed as an MIC change and assigned to one of the following three categories: No adaptive response (no MIC increase), weak adaptive response (MIC increase ≤ 4fold) and strong adaptive response (MIC increase > 4fold). For some bacterial species two or more studies were found resulting in data from various isolates or strains. That is why some bacterial species can be found in two or three categories depending on the results obtained with the various isolates or strains of the same species.

Adaptive bacterial response in Gram-negative species

No adaptive response was found in isolates or strains of 15 species (Acinetobacter baumannii, Aeromonas hydrophila, Campylobacter coli, Campylobacter jejuni, Chryseobacterium indologenes, Citrobacter spp., Cronobacter sakazakii, E. coli, K. pneumoniae, Moraxella osloensis, P. aeruginosa, Pseudomonas nitroreductans, Pseudomonas putida, Pseudoxanthomonas spp. and Sphingobacterium multivorum). Some isolates or strains of 12 species were able to express a weak adaptive response (MIC increase ≤ 4fold) such as A. xylosoxidans, A. jandaei, Chrysobacterium spp., E. cloacae, Enterobacter spp., E. coli, H. gallinarum, K. pneumoniae, P. aeruginosa, S. Typhimurium, Serratia spp. and S. maltophilia (Table 1).

TABLE 1:

Adaptive response of Gram-negative bacterial species to sublethal CHG exposure, adapted from [35].

Species Strain / isolate Type of exposure Increase in MIC MICmax (mg/l) Stability Associated changes Ref
A. xylosoxidans Domestic drain biofilm isolate MBRG 4.31 14 d at various concentrations 2fold 31.2 No data None reported [36]
A. baumannii Strain MBRG15.1 from a domestic kitchen drain biofilm 14 passages at various concentrations None 7.8 Not applicable None reported [37]
A. baylyi Strain ADP1 30 min at 0.000001% Protection from lethal CHG concentration (0.00007%) No data No data More resistance to a lethal hydrogen peroxide concentration (300 mM) [38]
A. hydrophila Domestic drain biofilm isolate MBRG 4.3 14 d at various concentrations None 15.6 Not applicable None reported [36]
A. jandaei Domestic drain biofilm isolate MBRG 9.11 14 d at various concentrations 2fold 15.6 No data None reported [36]
A. proteolyticus Domestic drain biofilm isolate MBRG 9.12 14 d at various concentrations 16fold 125 No data None reported [36]
B. fragilis ATCC 25285 12 h at 0.06% No data No data Not applicable Induction of multiple antibiotic resistance*; 2.7fold – 6fold increase of 6 efflux pumps [39]
B. cenocepacia 6 strains from clinical and environmental habitats Up to 28 d at 15 mg/l Survival 100 No data No degradation of CHG [40]
B. cepacia ATCC BAA-245 40 d at various concentrations 8fold 29 Unstable for 14 d Decrease biofilm formation [41]
B. cepacia complex B. lata strain 383 5 min at 50 mg/l No data 700 Not applicable Reduced susceptibility** to ceftazidime (30 – 33 mm), ciprofloxacin (11 – 20 mm) and imipenem (15 – 21 mm; 2 of 4 experiments) and to meropenem (33 mm; 1 of 4 experiments); up-regulation of transporter and efflux pump genes [42]
C. coli ATCC 33559 and a poultry isolate Up to 15 passages with gradually higher concentrations None 0.031 Not applicable None described [15]
C. jejuni NCTC 11168, ATCC 33560 and a poultry isolate Up to 15 passages with gradually higher concentrations None 1 Not applicable None described [15]
C. indologenes MRBG 4.29 (kitchen drain biofilm isolate) 40 d at various concentrations None 7.3 Not applicable None described [41]
C. indologenes Domestic drain biofilm isolate MBRG 9.15 14 d at various concentrations None 31.2 Not applicable None reported [36]
Chrysobacterium spp. Domestic drain biofilm isolate MBRG 9.17 14 d at various concentrations 2fold 7.8 No data None reported [36]
Chrysobacterium spp. 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 5fold – 6fold 30 Unstable Cross-adaptation* to benzalkoniumchloride (2fold - 100fold; 2 strains), triclosan (4fold; 1 strain) and didecyldimethyl- ammonium bromide (16fold; 1 strain); cross-resistance* to cefotaxime and ceftazidime (2 strains each), sulfamethoxazole, ampicillin and tetracycline (1 strain each) [43]
Citrobacter spp. Domestic drain biofilm isolate MBRG 9.18 14 d at various concentrations None 1.9 Not applicable None reported [36]
C. sakazakii Strain MBRG15.5 from a domestic kitchen drain biofilm 14 passages at various concentrations None 7.8 Not applicable None reported [37]
E. cloacae 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 10fold – 16fold 80 Stable for 20 subcultures (1 strain) Cross-adaptation* to benzalkoniumchloride (6fold; 2 strains), triclosan (6fold - 15fold; 2 strains) and didecyldimethylammonium bromide (6fold; 1 strain); cross-resistance* to imipenem, ceftazidime and sulfamethoxazole (2 strains each), cefotaxime and tetracycline (1 strain each) [43]
E. ludwigii 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 6fold – 8fold 40 Unstable Cross-adaptation* to benzalkoniumchloride (6fold – 8fold; 2 strains), triclosan (8fold – 10fold; 2 strains) and didecyldimethylammonium bromide (4fold – 6fold; 2 strains); cross-resistance* to imipenem, ceftazidime and sulfamethoxazole (2 strains each) [43]
Enterobacter spp. 6 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 4fold – 10fold 80 Stable for 20 subcultures (1 strain) Cross-adaptation* to benzalkoniumchloride (3fold – 20fold; 6 strains), triclosan (4fold – 100fold; 6 strains) and didecyldimethylammonium bromide (4fold – 6fold; 3 strains); cross-resistance* to ceftazidime and imipenem (3 strains each), cefotaxime and sulfamethoxazole (2 strains each) [43]
E. coli ATCC 25922 40 d at various concentrations None 7.3 Not applicable None described [41]
E. coli NCIMB 8879 6 x 48 h at variable concentrations None 0.7 Not applicable None reported [44]
E. coli ATCC 25922 and strain MBRG15.4 from a domestic kitchen drain biofilm 14 passages at various concentrations 1.5fold - 5fold 11.7 Stable for 14 d None reported [37]
E. coli NCIMB 8545 0.00005% for 30 s, 5 min and 24 h ≤ 6fold 39 Unstable for 10 d No increase of MBC; unstable resistance** to tobramycin [45]
E. coli NCTC 8196 12 w at various concentrations 32fold No data No data None described [46]
E. coli NCTC 12900 strain O157 6 passages at variable concentrations Approx. 500fold Approx. 500 Stable for 30 d Increased tolerance** to triclosan (15 mm) [47]
E. coli CV601 24.4 µg/l for 3 h No data 4.9 Not applicable Induction of horizontal gene transfer (sulfonamide resistance by conjugation) [48]
H. gallinarum Domestic drain biofilm isolate MBRG 4.27 14 d at various concentrations 2fold 31.2 No data None reported [36]
K. oxytoca 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 2fold – 8fold 40 Unstable Cross-adaptation* to benzalkoniumchloride (60fold; 1 strain), triclosan (3fold – 8fold; 2 strains) and didecyldimethyl- ammonium bromide (6fold; 1 strain) [43]
K. pneumoniae 7 “Murray isolates” from the pre-CHG era Up to 5 w at various concentrations None (5 isolates) 4fold (2 isolates) 256 Stable for 10 d None reported [49]
K. pneumoniae 7 modern isolates / strains Up to 5 w at various concentrations 4fold - 16fold > 512 Stable for 10 d None reported [49]
K. pneumoniae 6 clinical strains with a variety of antibiotic resistance markers 6 passages of 2 days at various concentrations 4fold – 16fold 512 Stable for 10 d Cross-resistance*** to colistin (6 strains); no cross-adaptation to benzalkoniumchloride, octenidine, hexadecylpyridinium chloride monohydrate and ethanol [16]
K. pneumoniae ATCC 13883 40 d at various concentrations 6.9fold 14.5 Stable for 14 d Increase biofilm formation [41]
Klebsiella spp. Biocide-sensitive strain from organic foods Several passages with gradually higher concentrations 2fold 30 Unstable Cross-adaptation* to benzalkoniumchloride (12fold) and triclosan (12fold); cross-resistance* to imipenem and ceftazidime [43]
M. osloensis Strain MBRG15.3 from a domestic kitchen drain biofilm 14 passages at various concentrations None 2.0 Not applicable None reported [37]
P. agglomerans 5 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 5fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (30fold – 40fold; 5 strains), triclosan (8fold – 100fold; 5 strains) and didecyldimethylammonium bromide (4fold - 6fold; 2 strains); cross-resistance* to cefotaxime and ceftazidime (3 strains each), tetracycline and sulfamethoxazole (2 strains each) and imipenem (1 strain) [43]
P. ananatis 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 10fold – 50fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (20fold – 30fold; 2 strains), triclosan (60fold – 100fold; 2 strains) and didecyldimethylammonium bromide (6fold; 2 strains); cross-resistance* to cefotaxime (2 strains), sulfamethoxazole, imipenem, ceftazidime and tetracycline (1 strain each) [43]
Pantoea spp. 3 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 5fold – 16fold 80 Unstable Cross-adaptation* to benzalkoniumchloride (6fold – 60fold; 2 strains), triclosan (8fold; 3 strains) and didecyldimethylammonium bromide (4fold - 6fold; 3 strains); cross-resistance* to tetracycline (2 strains), ampicillin, ceftazidime, cefotaxime, sulfamethoxazole and imipenem (1 strain each) [43]
P. aeruginosa 178 CHG sensitive strains Exposure to CHG None 625 Not applicable None reported [50]
P. aeruginosa ATCC 9027 40 d at various concentrations 2fold 14.5 Unstable for 14 d None described [41]
P. aeruginosa ATCC 9027 14 passages at various concentrations 4fold 31.3 Stable for 14 d None reported [37]
P. aeruginosa NCIMB 10421 6 x 48 h at variable concentrations 7fold 70 Stable for 15 d High MICs to BAC did not change in a relevant extent [44]
P. aeruginosa NCTC 6749 12 w at various concentrations 8fold – 32fold 1,024 Stable for 7 w None described [46]
P. nitroreductans Domestic drain biofilm isolate MBRG 4.6 14 d at various concentrations None 3.9 Not applicable None reported [36]
P. putida Strain MBRG15.2 from a domestic kitchen drain biofilm 14 passages at various concentrations None 7.8 Not applicable None reported [37]
Pseudomonas spp. Domestic drain biofilm isolate MBRG 9.14 14 d at various concentrations 16fold 15.6 No data None reported [36]
Pseudoxanthomonas spp. Domestic drain biofilm isolate MBRG 9.20 14 d at various concentrations None 0.97 Not applicable None reported [36]
Ralstonia spp. Domestic drain biofilm isolate MBRG 4.13 14 d at various concentrations 21fold 167 No data None reported [36]
S. Virchow Food isolate 6 passages at variable concentrations Approx. 120fold Approx. 120 Stable for 30 d Increased tolerance** to triclosan (0 mm) [47]
Salmonella enterica serovar Typhimurium Strain SL1344 5 min at 0.1, 0.5, 1 and 4 mg/l 13fold – 27fold 800 Unstable for 1 d 3fold – 67fold increase of tolerance*** to BAC [51]
Salmonella enterica serovar Typhimurium Strain 14028S 5 min at 1 and 5 mg/l 3fold – 33fold 1,000 Unstable for 1 d 2.5fold – 20fold increase of tolerance*** to BAC [51]
S. enteritidis ATCC 13076 7 d of sublethal exposure ≥ 10fold > 50 Unstable None reported [52]
Salmonella spp. 3 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 5fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (8fold – 30fold; 2 strains) and triclosan (4fold - 8fold; 3 strains) cross-resistance* to cefotaxime, nalidixic acid and imipenem (2 strains each), tetracycline and sulfamethoxazole (1 strain each) [43]
Salmonella spp. 6 strains with higher MICs to biocidal products 8 days at increasing concentrations 50fold – 200fold (2 strains) > 1,000 “stable” One strain with increased tolerance*** to tetracycline (> 16 mg/l), chloramphenicol (8 mg/l) and nalidixic acid (16 mg/l) [53]
S. marcescens Strain GSU 86-828 7 d exposure to CHG-containing contact lens solutions 8fold 50 No data Increased adherence to polyethylene [54]
S. marcescens ATCC 13880 40 d at various concentrations 9.6fold 116 Stable for 14 d Increase biofilm formation [41]
S. marcescens Clinical isolate 12 w at various concentrations 32fold – 128fold 2,048 Stable for 7 w None described [46]
Serratia spp. Not described 5 to 8 transfers “resistance“ to CHG No data “stable” None described [55]
S. multivorum Domestic drain biofilm isolate MBRG 9.19 14 d at various concentrations None 15.6 Not applicable None reported [36]
S. maltophilia Domestic drain biofilm isolate MBRG 9.13 14 d at various concentrations 4fold 62.5 No data None reported [36]
S. maltophilia MRBG 4.17 (kitchen drain biofilm isolate) 40 d at various concentrations 6fold 29 Stable for 14 d None described [41]
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*

spiral gradient endpoint method;

**

disc diffusion method;

***

broth microdilution;

****

macrodilution method

A strong but unstable MIC change (> 4fold) was found in isolates or strains of four species (Burkholderia cepacia, E. coli, Salmonella enteritidis, Salmonella Typhimurium). A strong and stable MIC change (> 4fold) was described for isolates or strains of seven species (E. coli, K. pneumoniae, P. aeruginosa, Salmonella Virchow, Salmonella spp., S. marcescens, Stenotrophomonas maltophilia). In isolates or strains of six species (Acinetobacter baylyi, Acinetobacter proteolyticus, E. coli, Pseudomonas spp., Ralstonia spp., S. marcescens) the adaptive response was strong but its stability was not described.

Selected strains or isolates revealed substantial MIC changes: E. coli (up to 500fold), Salmonella spp. (up to 200fold), S. marcescens (up to 128fold), P. aeruginosa (up to 32fold), or A. proteolyticus, K. pneumoniae, and Pseudomonas spp. (all up to 16fold). The highest MIC values after adaptation were found in S. marcescens (2,048 mg/l), P. aeruginosa (1,024 mg/l), Salmonella spp. (> 1,000 mg/l), B. cepacia complex (700 mg/l), K. pneumoniae (> 512 mg/l) and E. coli (500 mg/l). Most maximum MIC values are above the proposed epidemiological cut-off value of 16–64 mg/l to determine CHG resistance in Gram-negative bacterial species [12].

Cross resistance to various antibiotics such as tetracycline, gentamicin or meropeneme was found in some isolates of Bacterioides fragilis, B. cepacia complex and Salmonella spp.. In addition, a lower susceptibility to other biocidal agents was described for E. coli and S. Virchow to triclosan, for A. baylyi to hydrogen peroxide and for S. Typhimurium to benzalkonium chloride (BAC).

Other adaptive changes include a significant up-regulation of efflux pump genes in B. fragilis and B. cepacia complex. Horizontal gene transfer (sulfonamide resistance by conjugation) was induced in E. coli. VanA-type vancomycin resistance gene expression was increased vanA Enterococcus faecium (≥ 10fold increase of vanHAX encoding). Enhanced biofilm formation was described for K. pneumoniae and S. marcescens, adherence to poly-ethylene was increased in S. marcescens. Biofilm formation was decreased in B. cepacia.

Adaptive bacterial response in Gram-positive species

No adaptive response was found in isolates or strains from 18 species (Bacillus cereus, Corynebacterium xerosis, Enterococcus saccharolyticus, Eubacterium spp., Methylobacterium phyllosphaerae, Micrococcus luteus, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus kloosii, Staphylococcus lugdenensis, Staphylococcus saprophyticus, Staphylococcus warneri and Streptococcus mutans).

Some isolates or strains of 12 species ware able to express a weak adaptive response (MIC increase ≤ 4fold) such as B. cereus, Corynebacterium pseudogenitalum, Corynebacterium renale group, Enterococcus casseliflavus, Enterococcus faecalis, E. faecium, M. luteus, S. aureus, S. capitis, S. haemolyticus, S. lugdenensis and S. warneri.

A strong but unstable MIC change (> 4fold) was found in isolates or strains of E. faecalis. A strong MIC change (> 4fold) was also described for isolates or strains of S. aureus which could be stable or of unknown stability.

The largest MIC increase was noticed in S. aureus (up to 16fold) and E. faecalis (up to 6.7fold) leading to MIC values as high as 24.2 mg/l in E. faecalis and 20 mg/l in S. aureus (Table 2). Some maximum MIC values are above the proposed epidemiological cut-off value (8 mg/l for S. aureus) and some below (64 mg/l for E. faecalis) to determine CHG resistance in Gram-positive bacterial species [12].

TABLE 2:

Adaptive response of Gram-positive bacterial species to sublethal CHG exposure, adapted from [35].

Species Strain / isolate Type of exposure Increase in MIC MICmax (mg/l) Stability Associated changes Ref
B. cereus MRBG 4.21 (kitchen drain biofilm isolate) 40 d at various concentrations None 14.5 Not applicable None described [41]
B. cereus Domestic drain biofilm isolate MBRG 4.21 14 d at various concentrations None 1.9 Not applicable None reported [36]
B. cereus 4 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 6fold – 16fold 80 Stable for 20 subcultures (1 strain) Cross-adaptation* to benzalkoniumchloride (≥ 100fold; 3 strains), triclosan (4fold – 36fold; 3 strains) and didecyldimethylammonium bromide (6fold; 2 strains); cross-resistance* to imipenem (4 strains), sulfamethoxazole (2 strains), ampicillin and tetracycline (1 strain each) [43]
B. licheniformis 2 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 4fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (40fold - 75fold; 2 strains) and triclosan (8fold; 1 strain); cross-resistance* to imipenem (2 strains), cefotaxime and tetracycline (1 strain each) [43]
B. subtilis 2 strains and 3 derivates 2 h at 0.00005% No data No data Not applicable No increase of transfer of the mobile genetic element Tn916, a conjugative transposon [56]
Bacillus spp. 4 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 4fold – 8fold 40 Unstable Cross-adaptation* to benzalkoniumchloride (15fold – 100fold; 4 strains), triclosan (8fold; 4 strains) and didecyldimethylammonium bromide (4fold - 6fold; 2 strains); cross-resistance* to imipenem and sulfamethoxazole (4 strains each), cefotaxime and ceftazidime (1 strain each) [43]
C. pseudogenitalum Human skin isolate MBRG 9.24 14 d at various concentrations 4fold 3.9 No data None reported [36]
C. renale group Human skin isolate MBRG 9.13 14 d at various concentrations 4fold 31.2 No data None reported [36]
C. xerosis WIBG 1.2 (wound isolate) 40 d at various concentrations None 3.6 Not applicable None described [41]
E. casseliflavus 3 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 8fold – 20fold 100 Stable for 20 subcultures (1 strain) Cross-adaptation* to benzalkoniumchloride (30fold - 100fold; 4 strains), triclosan (> 100fold; 1 strain) and didecyldimethylammonium bromide (4fold - 6fold; 2 strains); cross-resistance* to imipenem (3 strains), cefotaxime and tetracycline (1 strain each) [43]
E. durans Biocide-sensitive strain from organic foods Several passages with gradually higher concentrations 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (≥ 100fold), triclosan (10fold) and didecyldimethylammonium bromide (16fold); cross-resistance* to imipenem and ampicillin [43]
E. faecalis 1 strain of unknown origin 14 passages at various concentrations 2fold 7.8 Stable for 14 d None reported [37]
E. faecalis Strain SS497 10 passages at various concentrations 3.7fold 11 Significant increase of surface hydrophobicity No data [57]
E. faecalis WIBG 1.1 (wound isolate) 40 d at various concentrations 6.7fold 24.2 Unstable for 14 d None described [41]
E. faecalis Biocide-sensitive strain from organic foods Several passages with gradually higher concentrations 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (80fold) and didecyldimethylammonium bromide (8fold); cross-resistance* to imipenem and ceftazidime [43]
E. faecium 9 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 2fold – 16fold 80 Stable for 20 subcultures (1 strain) Cross-adaptation* to benzalkoniumchloride (10fold - 100fold; 9 strains), triclosan (4fold - 100fold; 6 strains) and didecyldimethylammonium bromide (4fold - 8fold; 7 strains); cross-resistance* to imipenem (9 strains), tetracycline (4 strains), ampicillin (2 strains) cefotaxime and ceftazidime (1 strain each) [43]
E. faecium VRE strain 410 (skin and soft tissue infection isolate) 21 d at various concentrations 4fold 19.6 No data Subpolulation with reduced susceptibility* to daptomycin including significant alterations in membrane phospholipids [58]
E. faecium 3 vanA VRE strains 15 min at MIC No data No data Not applicable ≥ 10fold increase of vanHAX encoding VanA-type vancomycin resistance and of liaXYZ associated with reduced daptomycin susceptibility; vanA upregulation was not strain or species specific; VRE was more susceptible to vancomycin in the presence of subinhibitory chlorhexidine [59]
E. saccharolyticus Domestic drain biofilm isolate MBRG 9.16 14 d at various concentrations None 1.9 Not applicable None reported [36]
Enterococcus spp. 6 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 2fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (30fold - 100fold; 6 strains), triclosan (4fold - 15fold; 5 strains) and didecyldimethylammonium bromide (4fold - 6fold; 4 strains); cross-resistance* to imipenem (6 strains), ceftazidime and sulfamethoxazole (5 strains each), cefotaxime (4 strains), tetracycline (3 strains) and ampicillin (2 strains) [43]
Eubacterium spp. Domestic drain biofilm isolate MBRG 4.14 14 d at various concentrations None 31.2 Not applicable None reported [36]
M. phyllosphaerae Domestic drain biofilm isolate MBRG 4.30 14 d at various concentrations None 15.6 Not applicable None reported [36]
M. luteus MRBG 9.25 (skin isolate) 40 d at various concentrations None 3.6 Not applicable None described [41]
S. aureus ATCC 6538 40 d at various concentrations None 3.6 Not applicable None described [41]
S. aureus ATCC 6538 100 d at various concentrations None 0.6 Not applicable None described [60]
S. aureus NCTC 6571 plus 2 MRSA strains Several passages with gradually higher concentrations 1.3fold – 2fold 1 “unstable” None described [61]
S. aureus NCIMB 9518 0.00005% for 30 s, 5 min and 24 h 2fold – 5fold 20 Stable for 10 d No increase of MBC [45]
S. aureus ATCC 6538 7 d of sublethal exposure 2.5fold 2.5 Unstable for 10 d None reported [52]
S. aureus 3 clinical MRSA strains 10 passages at various concentrations ≤ 4fold 8 No data No change of PHMB susceptibility** [62]
S. aureus ATCC 6538 14 passages at various concentrations 4fold 7.8 Unstable for 14 d None reported [37]
S. aureus ATCC 25923 and 14 clinical isolates 14 d at various sublethal concentrations 4fold - 6fold (6 isolates) 6.3 No data Increased tolerance* to ciprofloxacin (4fold - 64fold; 10 isolates), tetracycline (4fold - 512fold; all isolates), gentamicin (4fold - 512fold; 8 isolates), amikacin (16fold - 512fold; 11 isolates), cefepime (8fold - 64fold; 11 isolates) and meropeneme (8fold - 64fold; 9 isolates) [63]
S. aureus NCTC 4163 12 w at various concentrations 16fold No data No data None described [46]
S. aureus Strain SAU3 carrying plasmid pWG613 10 min at 0.00005% No data No data Not applicable No significant reduction of plasmid transfer frequency [64]
S. capitis MRBG 9.34 (skin isolate) 40 d at various concentrations 1.7fold 6 Stable for 14 d None described [41]
S. capitis Human skin isolate MBRG 9.34 14 d at various concentrations None 7.8 Not applicable None reported [36]
S. caprae MRBG 9.3 (skin isolate) 40 d at various concentrations None 3.6 Not applicable None described [41]
S. caprae Human skin isolate MBRG 9.30 14 d at various concentrations None 7.8 No data None reported [36]
S. cohnii Human skin isolate MBRG 9.31 14 d at various concentrations None 3.9 Not applicable None reported [36]
S. epidermidis MRBG 9.33 (skin isolate) 40 d at various concentrations None 9.7 Not applicable None described [41]
S. epidermidis Human skin isolate M 9.33 14 d at various concentrations None 7.8 Not applicable None reported [36]
S. epidermidis CIP53124 1 d at various concentrations No data No data Not applicable Significant increase of biofilm formation at various sublethal concentrations [65]
S. haemolyticus Human skin isolate MBRG 9.35 14 d at various concentrations None 15.6 Not applicable None reported [36]
S. haemolyticus MRBG9.35 (skin isolate) 40 d at various concentrations 2.1fold 3 Unstable for 14 d None described [41]
S. hominis Human skin isolate MBRG 9.37 14 d at various concentrations None 7.8 Not applicable None reported [36]
S. kloosii Human skin isolate MBRG 9.37 14 d at various concentrations None 7.8 Not applicable None reported [36]
S. lugdunensis Human skin isolate MBRG 9.36 14 d at various concentrations None 15.6 Not applicable None reported [36]
S. lugdunensis MRBG 9.36 (skin isolate) 40 d at various concentrations 4fold 3.6 Stable for 14 d None described [41]
S. saprophyticus Human skin isolate MBRG 9.29 14 d at various concentrations None 3.9 Not applicable None reported [36]
S. saprophyticus 4 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 2fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (25fold - 100fold; 4 strains), triclosan (4fold - 8fold; 3 strains) and didecyldimethylammonium bromide (6fold - 12fold; 2 strains); cross-resistance* to ceftazidime (4 strains), imipenem, sulfamethoxazole and cefotaxime (2 strains each) and tetracycline (1 strain) [43]
S. warneri MRBG 9.27 (skin isolate) 40 d at various concentrations None 29 Not applicable None described [41]
S. warneri Human skin isolate MBRG 9.27 14 d at various concentrations 2fold 15.6 No data None reported [36]
S. xylosus Biocide-sensitive strain from organic foods Several passages with gradually higher concentrations 4fold 20 Unstable Cross-adaptation* to benzalkoniumchloride (> 100fold), triclosan (8fold) and didecyldimethylammonium bromide (20fold); cross-resistance* to ceftazidime, imipenem, sulfamethoxazole, cefotaxime and tetracycline [43]
Staphylococcus spp. 3 biocide-sensitive strains from organic foods Several passages with gradually higher concentrations 4fold – 10fold 50 Unstable Cross-adaptation* to benzalkoniumchloride (4fold - 10fold; 3 strains), triclosan (8fold - 100fold; 3 strains) and didecyldimethylammonium bromide (6fold - 20fold; 3 strains); cross-resistance* to ceftazidime (1 strain) [43]
S. mutans Strain UA159 10 passages at various concentrations None 3 Not applicable None reported [57]
Open in a new tab
*

broth microdilution;

**

macrodilution method

Cross tolerance to various antibiotics such as tetracycline, gentamicin or meropeneme could be found in some isolates of S. aureus. In E. faecium (vancomycin-resistant enterococcus; VRE) a more than 10fold vanA up-regulation was detected as well as reduced daptomycin susceptibility. An increase in biofilm formation was described in S. epidermidis.

DISCUSSION

The strongest adaptation to low level CHG exposure was found in common nosocomial pathogens such as E. coli (up to 500fold MIC increase), S. marcescens (up to 128fold MIC increase), P. aeruginosa (up to 32fold MIC increase) and K. pneumoniae (up to 16fold MIC increase). After sublethal exposure the highest MIC values were also found in common nosocomial pathogens such as S. marcescens (2,048 mg/l), P. aeruginosa (1,024 mg/l), K. pneumoniae (> 512 mg/l) and E. coli (500 mg/l), It is probably no coincidence that these pathogens are among those species considered to have extreme or even pan resistance to antibiotics [13].

Low level CHG exposure also reduced the susceptibility to selected antibiotics in Burkholderia spp. or Salmonella spp. In Burkholderia spp. an up-regulation of transporter and efflux pump genes was found. Efflux pumps are often not agent-specific and may well result in resistance to other biocidal agents or antibiotics [1]. A quite alarming finding was that horizontal gene transfer was induced in E. coli by low level CHG exposure enabling the faster spread of resistance genes within the bacterial community.

Some mechanisms of the adaptive response have been described. Increased expression of efflux pumps is recognized as a mechanism of antibiotic and biocide resistance. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps [14]. The multiple antibiotic resistance (mar) locus and mar regulon in E. coli and other members of the enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump, extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics [14]. In P. aeruginosa, a number of multidrug efflux pumps export a broad range of substrates [14]. In C. jejuni and C. coli active efflux was identified in adapted strains. In addition, the outer membrane protein profiles had changed, along with morphological changes [15]. In K. pneumoniae CHG adaptation was associated with mutations in the two-component regulator phoPQ and a putative Tet repressor gene (smvR) adjacent to the major facilitator superfamily (MFS) efflux pump gene, smvA [16]. And in Salmonella spp. a defense network was described that involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways. In addition, results indicated that CHG tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism [17].

A major limitation of this review is that most of the data were obtained in laboratories under defined conditions. The findings are certainly suitable to describe the potential for adaptation to CHG. But it is less clear if or how the findings are transferred to the clinic. In 2002 Block et al. described that the MIC for CHG was higher among clinical isolates when more CHG was used for any type of application [18]. A similar correlation between CHG usage and MIC values was described in 2018 with S. aureus [19]. Lindford et al. described an outbreak by MDR A. baumannii in a burn unit. One of the measures to finally control the outbreak was to reduce moist low-concentration CHG dressings on burn wounds [20]. And yet the clinical impact of an elevated MIC value remains under controversial debate [21]. In hand hygiene it is known that a low bactericidal effect of CHG on the skin can only be achieved in the presence of small volumes of water, the water released by the skin as transepidermal water loss does not seem to be sufficient [22]. If the water realised by the skin is sufficient to allow adaptive changes of the bacterial species on the skin is currently not know. And yet, the triclosan tale strongly suggested that “a chemical that constantly stresses bacteria to adapt, and behaviour that promotes antibiotic resistance needs to be stopped immediately when the benefits are null” [10]. CHG is obviously such a chemical that constantly stresses bacteria to adapt. Even if the clinical impact of isolates or strains with elevated MIC values cannot finally be evaluated at the moment it seems justified restricting the use of CHG to applications where health benefits are associated with its use.

IMPLICATIONS FOR HAND HYGIENE

Alcohol-based hand rubs

In alcohol-based hand rubs with additional CHG used for hygienic hand disinfection there is no sound evidence for an additional effect of CHG in vitro [23]. There is also no evidence on the prevention of any type of healthcare-associated infection by the additional CHG in hand rubs. But there are obvious risks such as acquired bacterial resistance, anaphylactic reactions or skin irritation [24]. Its use in the immediate patient environment may therefore contribute to the selection pressure especially when the CHG concentration is sublethal [20]. Additional biocidal agents in alcohol-based hand rubs such as CHG are not recommended by the WHO [7].

The same applies to hand rubs used for surgical hand disinfection [24]. For surgical hand disinfection additional biocidal agents such as CHG are not recommended because they do not contribute to the prevention of surgical site infections [3, 25]. Replacing hand rubs with additional CHG by hand rubs without CHG will help to reduce avoidable CHG selection pressure. They should, however, have an equivalent efficacy, dermal tolerance and user acceptability [26].

Antimicrobial soaps

Another simple option to reduce CHG selection pressure is to ban CHG soaps in healthcare for regular hand washing. Based on the WHO recommendation for hand hygiene from 2009 hand washing is recommended to wash hands when they are visibly soiled. The use of plain soap, however, is adequate, there is no health benefit for antimicrobial soaps [7].

Another possible use of antimicrobial soaps is prior to surgery. Surgical scrubbing usually lasts for 6–10 min of scrubbing time and consumes between 5 and 20 l water per scrub [27–29]. Surgical scrub products may only be effective with additional post-scrub water-based CHG treatments of the hands which pose an additional contamination and selection pressure risk [30, 31]. Alcohol-based hand rubs with an appropriate concentration of alcohol(s) have a stronger effect on the resident hand flora, require typically 1.5 min for application, cause less skin irritation [32] and do not pose any selection pressure to bacterial species due to their volatility [33, 34].

CONCLUSION

Overall, the evidence on the adaptive potential of various pathogens to low level CHG exposure strongly suggests to critically review the use of CHG in patient care and to eliminate it in all applications where no health benefit has been shown or is realistically expectable.

METHODS

A systematic literature search was conducted via the National Library of Medicine (PubMed) and via ScienceDirect (only research articles) on 10th March 2018 and up-dated on 25th June 2018 using the term chlorhexidine in combination with low level exposure (17 hits PubMed, 5 hits ScienceDirect), adaptive response (6/24), sublethal (27/72), resistance and MIC (142/640), and resistant and MIC (116/648). In addition, studies deemed suitable for this review were also included. Publications were included and results were extracted from them when they provided original data on any type of adaptive response to the exposure of bacteria to sublethal concentrations of CHG, corresponding changes of MICs (CHG, antibiotics, and other biocidal agents), survival in CHG solutions, efflux pump activity, gene expression or biofilm formation. Articles were excluded when they described only data on fungi, outbreaks, pseudo-outbreaks or infections caused by contaminated CHG products or solutions, only biochemical changes, an adaptive effect with other chlorhexidine salts or when a CHG solution or product was used for disinfection during an outbreak but without being the suspected or proven source. Reviews were also excluded and screened for any original information within the scope of the review.

The susceptibility of isolates or strains to CHG is described as the minimum inhibitory concentration (MIC value). In most studies it was described as a single value and is presented as such unless stated otherwise. The magnitude of any adaptive response to CHG is expressed as an MIC change and assigned to one of the following three categories: no adaptive response (no MIC increase), weak adaptive response (MIC increase ≤ 4fold) and strong adaptive response (MIC increase > 4fold).

SUPPLEMENTAL MATERIAL

All supplemental data for this article are available online at http://www.microbialcell.com/researcharticles/2019a-kampf-microbial-cell/.

Abbreviations:

BAC

– benzalkonium chloride,

CHG

– chlorhexidine digluconate,

MIC

– minimal inhibitory concentration,

PHMB

– polyhexanide.

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