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International Journal of Antimicrobial Agents 41 (2013) 65– 69
Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents
jou rn al h om epa ge: h t tp : / /www.e lsev ier .com/ locate / i jant imicag
hlorhexidine is a highly effective topical broad-spectrum agent againstandida spp.
. Salima,b, C. Moorec,d, N. Silikasa, J. Satterthwaitea, R. Rautemaac,d,e,∗
The University of Manchester, School of Dentistry, Higher Cambridge Street, Manchester M15 6FH, UKThe University of Jordan, School of Dentistry, Amman, JordanMycology Reference Centre Manchester, University Hospital of South Manchester, Manchester M23 9LT, UKThe University of Manchester, Faculty of Medical and Human Sciences, Institute of Inflammation and Repair, Manchester Academic Health Science Centre, Manchester M23 9LT, UKUniversity Hospital of South Manchester, Manchester M23 9LT, UK
r t i c l e i n f o
rticle history:eceived 13 March 2012ccepted 24 August 2012
eywords:luconazolehlorhexidineandidausceptibilityinimum inhibitory concentration
a b s t r a c t
The objective of this study was to compare the in vitro antifungal activities of chlorhexidine (CHX) andfluconazole (FLZ) against Candida isolates comprising eight different species associated with oral candi-dosis. A broth microdilution method as described in Clinical and Laboratory Standards Institute (CLSI)protocol M27-A3 was used to determine susceptibility. A total of 79 clinical isolates and reference strainsbelonging to eight different Candida spp. was tested. The minimum inhibitory concentration (MIC) wasthe lowest drug concentration that reduced growth by 50% for FLZ at 48 h and by 80% for CHX at 24 h and48 h. The geometric mean MIC (and MIC range) at 48 h for CHX was 3.03 mg/L (0.78–6.25 mg/L) and for FLZwas 19.12 mg/L (≤0.125–256 mg/L). Of the 79 isolates, 14 (18%) were resistant to FLZ (MIC ≥ 64 mg/L).All isolates were effectively inhibited by ≤6.25 mg/L CHX, and Candida CHX MICs are below the CHX
levels found in saliva following normal dosing. No cross-resistance between CHX and FLZ was detected(rs = 0.039, P = 0.733). CLSI M27-A3 methodology proved to provide reproducible results with clear end-points for CHX. In conclusion, the findings showed that CHX has excellent broad-spectrum antifungalactivity in vitro. It was effective at concentrations detected in saliva when using standard dosing regi-mens. Moreover, no cross-resistance was detected between CHX and FLZ, even among Candida spp. highlylsevie
resistant to FLZ.© 2012 E
. Introduction
Being the disease of the diseased, oral candidosis predominantlyffects immunosuppressed and medically compromised patients1]. In these high-risk patients, the oral cavity may provide aource for Candida causing systemic infection [2]. Oral candido-is has become a significant challenge in patients with persistingisk factors and a recurrent need for antifungal treatment [2].n particular, repeated courses of fluconazole (FLZ) have beenhown to constitute a risk for persistent colonisation with micro-iologically and clinically resistant Candida [2]. Oral candidosis
s a mixed multispecies candidal–bacterial biofilm infection thatrovides multiple challenges for its management [3]. The biofilm
ifestyle is commonly associated with poor drug penetration andntimicrobial recalcitrance as well as a risk of development ofesistance [3,4].
∗ Corresponding author. Present address: Education & Research Centre, 2nd Floor,ythenshawe Hospital, Southmoor Road, Manchester M23 9LT, UK.
el.: +44 161 291 5914; fax: +44 161 291 5806.E-mail address: [email protected] (R. Rautemaa).
924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemttp://dx.doi.org/10.1016/j.ijantimicag.2012.08.014
r B.V. and the International Society of Chemotherapy. All rights reserved.
A number of antifungal agents are available for the manage-ment of fungal infections [5]; however, the choice of antifungalssuitable for the treatment of oral candidosis is limited [2]. FLZ is awidely used systemic antifungal agent that is well tolerated, withlow toxicity and mild side effects [5], although in elderly patientswith reduced saliva production there is a risk of low drug levels inthe oral cavity and the emergence of resistance [6].
Moreover, non-albicans Candida spp. such as Candida glabrataand Candida krusei are intrinsically resistant to FLZ and are com-mon causes of oral candidosis [1,7,8]. In addition, its penetrationinto candidal biofilm is poor, leading to low drug concentrations,which again has a potential risk for selection and development ofresistant strains [9]. Nystatin is a highly effective topical antifungalwith few drug interactions. However, its four times daily dosage is asignificant challenge for patient compliance [10,11]. Echinocandinsare highly effective agents against Candida and Candida biofilms[12]. However, their availability only as an intravenous formula-tion and their high cost negate their use for the treatment of oral
candidosis [2].Chlorhexidine (CHX) has been used as an adjunctive therapeu-tic option for topical use owing to its broad-spectrum antimicrobialefficiency [13]. It is effective at low concentrations and has unique
otherapy. All rights reserved.
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broth microdilution series is presented in Table 2A and B, respec-tively. CHX demonstrated antifungal activity against all testedisolates, with low MICs ranging from 0.78 mg/L to 6.25 mg/L; trail-ing endpoints with CHX were usually not encountered and 100%
Table 1Chlorhexidine geometric mean minimum inhibitory concentration (MIC) resultsfor 79 Candida spp. isolates belonging to eight different species at 24 h and 48 hincubation.
Candida spp. (No. of isolates) MIC (mg/L) P-valuea
24 h 48 h
C. albicans (32) 4.05 5.03 0.001C. glabrata (13) 3.13 4.78 0.005C. dubliniensis (10) 3.13 3.13 1.0C. parapsilosis (6) 1.56 3.13 0.014C. guilliermondii (6) 0.78 1.10 0.046C. tropicalis (6) 2.78 3.13 0.317
6 N. Salim et al. / International Journa
ubstantivity extending its therapeutic effect in the oral cavity14,15] owing to its high adsorption capacity such that it can beetained in the oral cavity for long periods (up to 12 h) [14]. Con-equently, less frequent dosing can be used [14,16]. The mode ofction of CHX on Candida is still unclear but it has been suggestedhat it inhibits cell wall synthesis by binding to negatively chargedroups in the candidal cell wall, followed by intracellular materialeakage and cell death (reviewed in [17]). It appears to inhibit can-idal replication and the adhesion of Candida to epithelial cells andenture surfaces, all being crucial prerequisites for fungal infection14]. CHX has been described to have significant activity against C.lbicans in vitro, but less data exist for Candida spp. other than C.lbicans, such as C. glabrata, Candida tropicalis and C. krusei [13,17].t has also been shown to have superior efficacy against Candidaiofilms compared with FLZ in vitro and in vivo [13,18–21]. Fur-hermore, it can be used to impregnate denture liners to act as aong-term self-release drug carrier [22].
There is a clear clinical and microbiological need for evaluationf the in vitro antifungal activity of CHX. The present study aimed tonvestigate the antifungal activity of CHX against a panel of isolateselonging to a number of different Candida spp. commonly isolatedrom patients with oral candidosis and to compare its activity withhat of FLZ. The null hypotheses were: first, that CHX is effectivegainst a broad spectrum of Candida spp.; and second, that it has aomparable activity to FLZ at levels seen in saliva.
. Materials and methods
.1. Organisms and media
A total of 79 Candida isolates belonging to eight differentpecies, comprising 76 clinical isolates and 3 reference strains,ere tested against CHX and FLZ. Clinical isolates were obtained
rom the culture collection of the Mycology Reference Centreanchester (UK) and were predominantly obtained from muco-
utaneous and haematogenous sources from patients, includinghose with immunodeficiency, candidaemia and tissue-invasiveisease. American Type Culture Collection (ATCC) strains C. albicansTCC 90028, C. krusei ATCC 6258 and C. tropicalis ATCC 750ere used as reference strains [23]. Isolates were identified by
tandard biochemical methods, including CHROMagarTM Candidaedium (CHROMagar, Paris, France), API ID32C (bioMérieux, Lyon,
rance) assimilation tests, and Candida dubliniensis agglutinationest (Bichro-Dubli Fumouze®; Fumouze Diagnostics, Levallois-erret, France). Isolates were stored at −80 ◦C and each isolateas plated twice on Sabouraud agar (Oxoid Ltd., Basingstoke, UK)
nd incubated at 37 ◦C for 48 h before use to check viability andurity. A total of 32 C. albicans, 13 C. glabrata, 10 C. dublinien-is, 6 Candida parapsilosis, 6 Candida guilliermondii, 6 C. tropicalis,
C. krusei and 1 Candida kefyr were tested. RPMI-1640 with 2%lucose, buffered with morpholinopropanesulfonic acid (MOPS)Sigma-Aldrich, Dorset, UK) and adjusted to pH 7.0 was used asrowth medium for FLZ and CHX.
The reproducibility of the method was evaluated by retesting0% of randomly selected isolates (16/79) against each drug. Theame batch of medium was used throughout the study, includingeproducibility studies.
.2. Susceptibility testing and antifungal agents
The broth microdilution method as described in Clinical and
aboratory Standards Institute (CLSI) document M27-A3 [24] wassed to determine susceptibility. FLZ (Pfizer, Sandwich, UK) andHX (Sigma-Aldrich) were obtained in pure powder form fromheir respective manufacturers. Briefly, a two-fold dilution seriestimicrobial Agents 41 (2013) 65– 69
of FLZ (0.125–2048 mg/L) and CHX (0.1–50 mg/L) was prepared insterile distilled water, and an inoculum of 1 × 103 organisms/mLwas used. Following incubation at 37 ◦C, growth in each well wasdetermined by measuring the optical density at 490 nm (OD490)with a spectrophotometer (BMG Labtech, Aylesbury, UK). For FLZ,the minimum inhibitory concentration (MIC) was the lowest drugconcentration that reduced the OD490 by 50% at 48 h compared withthe drug-free control. CLSI standard breakpoints for FLZ were usedfor susceptibility interpretation [23,25]. Isolates were designatedsusceptible, susceptible dose-dependent or resistant based on theirMICs and according to CLSI standards [23,25]. For CHX, the MIC wasthe lowest drug concentration that reduced the OD490 by 80% at 24 hand 48 h compared with the drug-free control [23]. Sabouraud agarand blood agar plates were inoculated with 10 �L of each organismsuspension to check the viable count and culture purity. Geometricmeans (GMs) and ranges were calculated.
2.3. Statistical analysis
SPSS statistical package v.18.0 (SPSS Inc., Chicago, IL) was usedto analyse all data. Kruskal–Wallis test was used to verify differ-ences in susceptibility between species against CHX at P < 0.05 withpost hoc Mann–Whitney U-tests. A Wilcoxon test was performedto compare MICs at 24 h and 48 h for each species against CHX. Thecorrelation between the antifungal activity of CHX and FLZ againstCandida spp. was evaluated using Spearman’s rho (rs) coefficient.The ranking was used to establish whether this correlation coeffi-cient is significantly different from zero. The significance level wasdetermined at P ≤ 0.05.
3. Results
The GM MIC for CHX for all Candida isolates was 2.22 mg/L at24 h and 3.03 mg/L at 48 h (Table 1) and the MIC90 (MIC for 90% ofthe organisms) was 6.25 mg/L (range 0.78–6.25 mg/L) at 48 h. TheMIC at 48 h was significantly higher than at 24 h for five species(C. albicans, C. glabrata, C. parapsilosis, C. guilliermondii and C. kru-sei) (P ≤ 0.05). For three species (C. dubliniensis, C. tropicalis andC. kefyr), the incubation time did not have an impact on the MIC(P > 0.05), although only a small number of isolates were tested forsome species. The GM MIC for C. albicans and C. glabrata was sig-nificantly higher than that detected for all other species at 48 h(P < 0.05).
The cumulative percentage of isolates for each species of Can-dida inhibited at each concentration of CHX and FLZ throughout the
C. krusei (5) 1.56 3.13 0.025C. kefyr (1) 0.78 0.78All isolates (79) 2.22 3.03
a Differences between MICs at 24 h and 48 h were tested by Wilcoxon test.
N. Salim et al. / International Journal of Antimicrobial Agents 41 (2013) 65– 69 67
Table 2Cumulative percentage of isolates for each species of Candida inhibited at each concentration in broth microdilution series.
Candida spp. (No. of isolates) Cumulative percentage of strains at MIC (mg/L)
≤0.09 0.19 0.39 0.78 1.56 3.125 6.25 12.5 25 ≥50
(A) Susceptibility of Candida spp. to chlorhexidine by MICa at 80% after 48 hC. albicans (32) 31 100C. glabrata (13) 38 100C. dubliniensis (10) 100C. parapsilosis (6) 100C. guilliermondii (6) 33 100C. tropicalis (6) 16 100C. krusei (5) 100C. kefyr (1) 100All isolates (79) 4 9 62 100
Candida spp. (No. of isolates) Cumulative percentage of strains at MICb (mg/L)
≤0.125 0.25 0.5 1 2 4 8 16 32 64 >64
(B) Susceptibility of Candida spp. to fluconazole by MICa at 50% after 48 hC. albicans (32) 3 69 81 87 90 94 97 100C. glabrata (13) 15 38 77 100C. dubliniensis (10) 60 90 100C. parapsilosis (6) 33 67 83 100C. guilliermondii (6) 50 67 100C. tropicalis (6) 16 33 67 100C. krusei (5) 60 100C. kefyr (1) 100All isolates (79) 9 39 49 52 57 62 70 73 82 90 100
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a Broth microdilution MICs determined using Clinical and Laboratory Standards Ib MIC breakpoints according to CLSI standards [23,25]: susceptible, ≤8 mg/L; sus
f the tested isolates were inhibited by 6.25 mg/L CHX. For FLZ, theICs ranged from ≤0.125 mg/L to 256 mg/L with an overall GM MIC
f 19.12 mg/L and a MIC90 of 128 mg/L (range ≤0.125–256 mg/L). Ofll isolates, 18% were resistant to FLZ (MIC ≥ 64 mg/L) (Table 2B).LZ was highly active against C. dubliniensis, C. parapsilosis, C. guil-iermondii and C. kefyr (all had a MIC ≤ 8 mg/L) but was less activegainst C. krusei, C. glabrata and C. tropicalis (0%, 15% and 16%,espectively, had a MIC ≤ 8 mg/L). Of the C. albicans isolates, 90%ad a MIC ≤ 8 mg/L. Candida krusei was highly resistant (100% had aIC ≥ 64 mg/L). None of the FLZ-resistant isolates were resistant to
linically relevant levels of CHX (20–154 mg/L) and the MICs for FLZid not correlate with those for CHX (rs = 0.039, P = 0.733), wherebyo cross-resistance was detected in vitro (Fig. 1). The lack of corre-
ation of CHX and FLZ MICs is demonstrated in Fig. 1. The GM MIC
ig. 1. Scatter plot showing the relationship between fluconazole and chlorhexidineinimum inhibitory concentrations (MICs) obtained with 79 isolates of Candida spp.
rs = 0.039, P = 0.733). Each number in the graph represents the number of isolatesith a particular chlorhexidine MIC and the corresponding fluconazole MIC.
te (CLSI) protocol M27-A3 [24].le dose-dependent, 16–32 mg/L (grey shaded area); resistant, ≥64 mg/L.
of all Candida isolates for FLZ was greater than six-fold that of CHX,and CHX showed minimal variation: MICs for CHX were within fourdilutions whereas MICs for FLZ were within 12 dilutions (Fig. 1).
The MIC of all Candida isolates was lower than the trough CHXconcentration normally detected in saliva (20 mg/L) [26] using nor-mal dosing regimens (twice-daily rinsing) (Fig. 2). The peak salivaconcentration of FLZ when normal dosing regimens of a singleoral dose of 100 mg of FLZ are used (2.56 mg/L) [27] exceeds theMICs of 57% of all tested Candida isolates, 81% of C. albicans andnone of the C. glabrata isolates. The MIC of 48% of all Candidaisolates was higher than the trough FLZ concentration normallydetected in saliva (1.4 mg/L). This percentage represents 10% ofC. dubliniensis, 19% of C. albicans, 33% of C. parapsilosis, 84% ofC. tropicalis, 100% of C. krusei and 100% of C. glabrata isolatestested.
The MICs for C. albicans ATCC 90028, C. krusei ATCC 6258 andC. tropicalis ATCC 750 tested as part of quality control were withinthe recommended ranges [23]. All 16 isolates when retested againstFLZ and CHX produced either identical results or gave a result thatdiffered by only one two-fold dilution.
4. Discussion
The current study showed that CHX has excellent antifungalactivity in vitro against a broad range of Candida spp. It was effec-tive against all Candida spp. tested at concentrations detected insaliva when using standard dosing regimens. These findings are inline with previous limited data [28]. Susceptibility to CHX was com-pared with that of FLZ for a wide range of Candida spp. Of the testedisolates, 12% were susceptible dose-dependent and 18% were resis-tant to FLZ. Interestingly, CHX was effective against those Candidaisolates with reduced susceptibility to FLZ, and no cross-resistance
was detected. This has not been reported previously. Consequentlythe first hypothesis, that CHX is effective against a broad spec-trum of Candida spp., was accepted, but the second hypothesis wasrejected as its efficacy was found to be superior to that of FLZ.
68 N. Salim et al. / International Journal of An
Fig. 2. Distribution and geometric mean ( ) minimum inhibitory concentrations(MICs) of 79 Candida isolates comprising eight Candida spp. for (a) chlorhexidineand (b) fluconazole. The hatched area represents the normal salivary concentrationfs
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biofilms. J Antimicrob Chemother 2002;49:973–80.
or each agent with normal dosing regimens, i.e. chlorhexidine rinse twice daily andingle oral dose of 100 mg of fluconazole [26,27].
Candida albicans and C. glabrata appeared to be less susceptibleo CHX compared with the other species tested. Nevertheless, their
ICs were lower than the mean trough salivary CHX levels when atandard twice-daily rinsing regimen is used [26]. The highest MICecorded was 6.25 mg/L, and the mean level of CHX in saliva at 12 hfter rinsing has been reported to be 20 mg/L [26]. The highest MICsere also 10-fold lower than the breakpoint of 70 mg/L previously
uggested for oral bacteria [29]. These in vitro results are consis-ent with the results of a previous in vivo study that showed goodesponse to CHX treatment of oral candidosis [30].
FLZ showed considerable antifungal activity against 70% of theested isolates. However, some or all C. krusei, C. tropicalis and C.labrata showed resistance. FLZ breakpoints for C. albicans, C. tropi-alis, C. parapsilosis, C. glabrata and C. krusei applied in this study areresently subject to revision and suggested new breakpoints wouldesignate an even higher proportion of the isolates as resistant [31].
mportantly, the MICs of 43% of the tested Candida isolates, 19%
f C. albicans and 100% of C. glabrata were higher than the meaneak saliva concentrations of FLZ with a standard dosing regimenf once-daily oral 100 mg of FLZ [27]. At 24 h, the salivary level of[
[
timicrobial Agents 41 (2013) 65– 69
FLZ falls below the MICs of 48% of the tested isolates [27]. This isof clinical importance since the risk for development of antifungalresistance in Candida spp. has been linked to low drug levels [2].Moreover, even the highest achievable salivary FLZ concentrationsare lower than the MICs of all C. glabrata isolates. Candida glabrata isan important cause for oral candidosis and particularly for denturestomatitis [1].
There is no established in vitro method for testing the suscepti-bility of Candida to CHX. This study confirms that the CLSI M27-A3methodology is suitable and provides reproducible results withclear endpoints [24]. The results of this study also showed a signif-icant increase in MIC readings from 24 h to 48 h for most species.This is in line with the CLSI standard that generally recommends thefinal reading at 48 h for all drugs [23]. However, the CLSI standardprovides 24-h reading breakpoints for C. parapsilosis and C. krusei,whereas the current results show a significant increase for thesespecies after 24 h. On the other hand, in this study the differencebetween the two readings for C. dubliniensis and C. tropicalis wasnot significant. The relationship between in vitro MIC results andclinical outcome is complex [4]. A number of patient variables suchas the immune status of the patient and the chronicity of the fun-gal infection may affect the response, and the MIC alone and theMIC/tissue level ratio does not predict treatment success. For exam-ple, it is possible that FLZ levels reached in oral epithelium may besufficient to inhibit the invasion of Candida into underlying tissuesand ease the symptoms despite low salivary levels and no changein the candidal load in the oral cavity.
In conclusion, these findings demonstrate that CHX has excel-lent antifungal efficacy against a broad range of Candida spp. and issuperior to that of FLZ. It was effective at concentrations detected insaliva when using standard dosing regimens. Moreover, no cross-resistance was detected between CHX and FLZ, even among Candidaspp. highly resistant to FLZ. This work, together with the previousdata showing excellent activity of CHX against candidal biofilms,reinforces the use of CHX for the treatment of oral candidosis [13]. Itcould be used as an alternative to commonly used antifungal drugsor as an adjunct therapy especially in complicated recurrent oralfungal infections.
Funding: This work was supported in part by the University ofJordan (Amman, Jordan) (to NSa).
Competing interests: None declared.Ethical approval: Not required.
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