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The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds Monika Sienkiewicz a,*, Katarzyna Poznan´ska-Kurowska b,1, Andrzej Kaszuba b,1, Edward Kowalczyk c,2 a
Medical and Sanitary Microbiology Department, Medical University of Lodz, Lodz, Pl Hallera 1, 90-647, Poland Department of Dermatology, Pediatric Dermatology and Oncology, Medical University of Lodz, Kniaziewicza 1/5, 91347 Lodz, Poland c Pharmacology and Toxicology Department, Medical University of Lodz, Lodz, Pl. Hallera 1, 90-647, Poland b
article info
abstract
Article history:
Hard-to-heal wounds represent a significant problem to patients, health care professionals,
Accepted 7 November 2013
and health care system. They can be formed as a result of mechanical injuries and burns, and any co-existing chronic disease increases the risk of their emergence. Diabetics are at a
Keywords:
greater risk of developing chronic wounds because of poor circulation, slow healing times,
Geranium oil
vascular disease and neuropathy.
Inhibiting activity
The aim of this study was to determine the antimicrobial activity of geranium oil against Gram-negative bacterial clinical strains. Clinical strains were isolated from patients with
Wounds Gram-negative pathogens
difficult-to-treat wounds and a comprehensive evaluation of their sensitivity to antibiotics was carried out. The constituents of geranium oil were specified by GC–FID–MS analysis. The micro-dilution broth method was used to check the inhibition of microbial growth at various concentrations of geranium oil. The tested geranium oil was efficacious against Gramnegative pathogens responsible for problems with wound treatment. The results suggest that geranium oil may be considered an effective component of therapy in the case of frequent recurrences of infections caused by resistant pathogens. # 2013 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Hard-to-heal wounds and ulcers present serious health complications and are significant factors in the cost of hospital treatment. This problem is caused by the spread of resistance to antibiotics commonly used in clinical practice. Both Gram-positive bacteria such as Staphylococcus aureus, coagulase-negative staphylococci, beta-hemolytic Streptococcus
(S. pyogenes, S. agalactiae), and Gram-negative bacteria such as Escherichia coli, Enterobacter species, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter baumanii are pathogens of great importance in wound infections. Aerobic gram-negative rods also infect wounds late in the course of chronic wound degeneration. Pseudomonas, Acinetobacter, Stenotrophomonas are usually acquired from exogenous sources such as bathwater [1]. From the clinical point of view, significant resistant strains of staphylococci include Methicillin-Resistant
* Corresponding author. Tel.: +48 42 639 33 42; fax: +48 42 639 33 42. E-mail addresses: [email protected] (M. Sienkiewicz), [email protected] (K. Poznan´ska-Kurowska), [email protected] (E. Kowalczyk). 1
Tel.: +48 42 651 10 72; fax: +48 42 651 10 72. Tel.: +48 42 639 33 35; fax: +48 42 639 33 35. 0305-4179/$36.00 # 2013 Elsevier Ltd and ISBI. All rights reserved. http://dx.doi.org/10.1016/j.burns.2013.11.002 2
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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S. aureus – MRSA, S. epidermidis – MRSE and S. aureus strains with reduced susceptibility to vancomycin and strains of S. aureus resistant to vancomycin [2]. The presence of constitutive and inductive Macrolide–Lincosamide–Streptogramin B – MLSB resistance mechanisms in streptococci limits the therapeutic possibilities. M-phenotype related to mef(A) gene conditioning resistance to erythromycin and other macrolides [3]. ExtendedSpectrum Beta-Lactamases (ESBLs) – enzymes produced by Gram-negative pathogens as Escherichia, Enterobacter, Klebsiella hydrolyse all penicillin-based antibiotics, monobactams and cephalosporins, even those with a wide range of activity. Multiresistant P. aeruginosa and Acinetobacter are resistant to fluoroquinolones, extended-spectrum cephalosporins, carbapenems and aminoglycosides. Clinical strains of Pseudomonas and Proteus produce metallo-beta-lactamase enzymes (Znblactamase) responsible for resistance to carbapenems [4–6]. Many kinds of dressings are used in wound treatment: absorbent, nonadherent, occlusive and semiocclusive, hydrophilic and hydrophobic, hydrocolloid and hydrogel, as well as alginates and absorbable materials, many of which could be a carrier for the testing of new, more effective antimicrobial drugs. The increasing presence of antibiotic-resistant pathogens strains has aroused interest in topical antimicrobial agents. Wound dressings containing silver have broad spectrum antimicrobial activity that encompasses many multidrug resistant wound pathogens. Jones et al. report that dressings with silver have great antibacterial effectiveness against methicillin-resistant S. aureus (MRSA) and P. aeruginosa [7,8]. Edwards-Jones et al. demonstrate that geranium essential oil vapors have strong antibacterial effects when used alone, against methicillin-resistant S. aureus when used in combination with grapefruit seed extract, and against methicillin-sensitive S. aureus when used with tea tree oil [9]. Essential oils can also be of huge relevance in Dermatology, especially in the healing of wounds and burns thanks to their antibacterial, antiviral and antifungal activities. In addition, most have anti-inflammatory, antioxidant and anti-tumor properties [10–13]. For example, according to Maruyama et al. the cutaneous application of several essential oils, especially geranium oil, may suppress the inflammatory symptoms related to neutrophil accumulation and edema [14]. The aim of our study was to determine the antibacterial activity of geranium oil against five genera of Gram-negative clinical isolates from patients with difficult-to-treat wound infections.
2.
Materials and methods
2.1.
Bacterial strains and their identification
The samples of Gram-negative clinical strains were isolated from the swabs of patients in the Clinic of Dermatology, Pediatric Dermatology and Oncology, Medical University of Lodz, Poland in 2012. The study included 63 patients, 38 males and 25 females, with a mean age of 46–58 years, All had wounds arising during the course of diabetes or non-healing wounds after burns. Gram-negative clinical strains isolated from wounds were cultured according to standard microbiological methods with use of Columbia Agar (bioMerieux) with
5% blood and Mac Conkey Agar (bioMerieux). They were identified to the species with use of API 20 E and API 20 NE tests (bioMerieux) according to manufacturer’s instructions. The bacteria were incubated at 37 8C for 24 h.
2.2.
Preparation and GC/FID/MS analysis of essential oils
A commercial essential oil from Pelargonium graveolens Ait. was purchased from the manufacturer (POLLENA-AROMA Poland) and analyzed by GC–FID–MS in the Institute of General Food Chemistry, Lodz University of Technology, using a Trace GC Ultra apparatus (Thermo Electron Corporation) with FID and MS DSQ II detectors and FIDMS splitter (SGE). Operating conditions: apolar capillary column Rtx-1ms (Restek), 60 m 0.25 mm i.d., film thickness 0.25 mm; temperature program, 50–300 8C at 4 8C/min; SSL injector temperature 280 8C; FID temperature 300 8C; split ratio 1:20; carrier gas helium at a regular pressure 200 kPa; FID temperature 260 8C; carrier gas, helium; 0.5 mL/min; split ratio 1:20. Mass spectra were acquired over the mass range 30–400 Da, ionization voltage 70 eV; ion source temperature 200 8C. The components were identified by comparing their mass spectra with those from a laboratory-made MS library, commercial libraries (NIST 98.1, Wiley Registry of Mass Spectral Data, 8th Ed. and MassFinder 4) and with literature data [15,16] along with the retention indices on an apolar column (Rtx-1, MassFinder 4) associated with a series of alkanes with linear interpolation (C8–C26).
3.
Antimicrobial activity testing
3.1.
Susceptibility testing
The tested strains were cultivated on Columbia Agar medium and incubated at 37 8C for 24 h. Bacterial suspensions with an optical density of 0.5MF were prepared with the bioMerieux densitometer, spread on Mueller-Hinton II Agar and incubated at 37 8C for 18 h. Susceptibility testing was carried out with the use of the disk-diffusion method, the following antibiotics (Becton Dickinson) being used against all tested E. coli, Citrobacter freundii, Enterobacter sakazakii, Enterobacter cloacae, P. aeruginosa and P. mirabilis clinical strains: GM – gentamicin (10 mg), PIP – piperacillin (100 mg), TIC – ticarcillin (75 mg), TZP – piperacillin/tazobaktam (100/10 mg), TIM – ticarcillin/clavulanic acid (75 mg/10 mg), CTX – cefotaxim (30 mg), CAZ – ceftazidime (30 mg), FEP–cefepim (30 mg), ATM–aztreonam (30 mg), IPM – imipenem (10 mg), MEM – meropenem (10 mg), ETP – ertapenem (10 mg), DOR – doripenem (10 mg), CIP – ciprofloxacin (5 mg), AN – amikacin (30 mg), NET – netilmicin (30 mg), TOB – tobramycin (10 mg). For clinical strains of E. coli, C. freundii, E. sakazakii and E. cloacae: CXM – cefuroxime (30 mg), C – chloramphenicol (30 mg), TE – tetracycline (30 mg), TGC – tigecyclin (15 mg), SXT – trimethoprim/sulfamethoxazole (1.25 mg/23.75 mg), only for E. coli strains: AMC – amoxicillin/ clavulanic acid (20 mg/10 mg), CF – cephalothin (30 mg), CZ – cefazolin (30 mg), AM – ampicillin (10 mg), FOX – cefoxitin (30 mg) and only for P. aeruginosa and P. mirabilis strains: LVX – levofloxacin (5 mg), CL – colistin (50 mg) additionally being used.
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [17].
3.2.
Minimal inhibitory concentration (MIC) assay
The antibacterial activity of the tested oil was investigated by the micro-dilution broth method. The essential oil was diluted in ethanol. This stock solution was mixed with a 100 ml Mueller-Hinton broth to obtain concentrations from 2.0 ml/ml to 9.0 ml/ml. An inoculum containing 1.5 108 CFU (10 ml) per well was added to broth with various oil concentrations; one well was filled with broth without oil to act as a control for strain growth. The mixtures were then transferred to 96-well microtiter plates. The Minimal Inhibitory Concentration (MIC) was determined as the lowest concentration of oil which inhibits the visible growth of bacteria after 24 h of incubation at 37 8C under aerobic conditions. The control media containing only alcohol at concentrations used in the dilutions of geranium oil did not inhibit the growth of bacterial strains.
4.
Results
4.1.
Tested Gram-negative bacterial strains
Sixty-three samples of wound swabs from patients were examined in the present study. Seventy five clinical strains of bacteria were isolated from swabs, among them were E. coli (n = 14, 18.6%), C. freundii (n = 6, 8.0%), E. sakazakii (n = 8, 10.6%), E. cloacae (n = 19, 25.3%), P. mirabilis (n = 11, 14.6%) and P. aeruginosa (n = 17, 22.6%).
4.2.
Constituents of the tested geranium oil
The results of the tested geranium oil analysis revealed that the main components were citronellol (26.7%) and geraniol (13.4%). Among sixty-one constituents identified in the geranium oil, the prevalent compounds included nerol (8.7%), citronellyl formate (7.1%), isomenthone (6.3%), linalool (5.2%), 10-epi-geudesmol (4.4%), geranyl formate (2.5%), menthone (1.6%), bcaryophyllene (1.5%), geranyl butyrate (1.4%), cis-rose oxide (1.4%), geranial (1.1%), and b-baurobonene (1.1%). The constituents of the tested oil are shown in Table 1.
4.3.
Bacterial susceptibility testing to antibiotics
The number of resistant strains to recommended antibiotics of the seventy-five clinical strains of E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa are shown in Table 2. The results show that the strains isolated from difficult-to-treat wound infections in the study were highly resistant to most of the b-lactam, cephalosporin, aminoglycoside and quinolone antibiotics recommended for susceptibility testing. Most strains were resistant to b-lactam antibiotics with inhibitors: amoxicillin with clavulanic acid, piperacyllin with tazobaktam and tikarcillin with clavulanic acid, which were used according to Clinical and Laboratory Standards Institute recommendations. Some of the E. coli, E. cloacae, P. mirabilis and P. aeruginosa strains isolated were resistant to
Table 1 – The main constituents of geranium essential oil. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Compound
%
RI
a-Pinene Limonene cis-Linlool oxide (f) Linalool cis-Rose oxide trans-Rose oxide Menthone Isomenthone a-Terpineol Citronellol Nerol Geraniol Geranial Citronellyl formate Geranyl formate Bicycloelemene Citronellyl acetate a-Cubebene Geranyl acetate a-Copaene b-Bourbonene 1,5-di-epi-Bourbonene b-Caryophyllene Citronellyl propionate b-Copaene Guaia-6,9-diene 4bH,10aH-Guaia-1(5),6-diene Geranyl propionate Alloaromadendrene 7aH,10bH-Cadina-1(6),4-diene Germacrene D b-Selinene Bicyclogermacrene a-Muurolene g-Cadinene trans-Calamenene d-Cadinene Zonarene Selina-4(15),7(11)-diene Geranyl butyrate Phenylethyl tiglate 10-epi-g-Eudesmol Geranyl tiglate Geranyl ester I
0.7 0.2 0.3 5.2 1.4 0.6 1.6 6.3 0.3 26.7 8.7 13.4 1.1 7.1 2.5 0.4
929 1021 1058 1086 1097 1113 1133 1144 1173 1217 1220 1243 1246 1261 1283 1334
0.2 0.4 0.5 1.1 0.2 1.5 0.3 0.2 0.3 0.5 1.0 0.2 0.2 1.0 0.2 0.7 0.2 0.6 0.3 0.9 0.2 0.2 1.4 0.7 4.4 1.0 0.2
1349 1361 1377 1385 1388 1419 1425 1428 1439 1445 1452 1459 1469 1477 1482 1491 1496 1509 1510 1515 1518 1530 1537 1554 1613 1675 1694
% Percentage of constituents; RI, Retention Index. The content of eighteen remaining constituents: Myrcene, a-Phellandrene, pCymene, (E)-b-Ocimene, trans-Linlool oxide (f), Isopulegol, Isomenthol, Estragole, Neryl formate, a-Gurjunene, 4aH,10aH-Guaia1(5),6-diene, g-Muurolene, g-Selinene, Dihydroagarofuran, Cadina1,4-diene, Geranyl isovalerate, g-Eudesmol, and Geranyl ester II amounted to 0.1%.
monobactam – aztreoname. The tested strains were generally sensitive to carbapenems, but few of E. coli strains were resistant to meropenem. Most of P. aeruginosa and P. mirabilis clinical isolates were resistant to carbapenems: meropenem and imipenem.
4.4.
Gram-negative bacteria susceptibility to geranium oil
Geranium essential oil demonstrated the greatest antibacterial activity against E. coli clinical strains isolated from wound
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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Table 2 – Antibiotic resistance demonstrated by Gram-negative clinical bacterial strains isolated from wound swabs. Tested Gram-negative bacteria E. coli n = 14
Resistance to tested antibiotics
GM(10); PIP(10); TIC(11); TZP(6); TIM(9), CTX(8); CAZ(7); FEP(5); ATM(3); IPM(0); MEM(3); ETP(0); DOR(0); CIP(3); AN(7); NET(8); TOB(2); CXM(2); C(10); TE(13); TGC(3); SXT(8); AmC(11); CF(4); CZ(4); AM(14); FOX(8); GM(3); PIP(3); TIC(3); TZP(4); TIM(3); CTX(4); CAZ(0); FEP(0); ATM(0); IPM(0); MEM(0); ETP(0); DOR(0); CIP(2); AN(4); NET(2); TOB(5); CXM(6); C(6); TE(6); TGC(0); SXT(3) GM(7); PIP(0); TIC(8); TZP(4); TIM(8); CTX(0); CAZ(0); FEP(0); ATM(0); IPM(0); MEM(0); ETP(0); DOR(0); CIP(4); AN(8); NET(8); TOB(4); CXM(7); C(6); TE(4); TGC(2); SXT(8) GM(19); PIP(4); TIC(15); TZP(3); TIM(12); CTX(6); CAZ(4); FEP(3); ATM(4); IPM(0); MEM(0); ETP(0); DOR(0); CIP(5); AN(9); NET(16); TOB(10); CXM(14); C(16); TE(7); TGC(5); SXT(15) GM(12); PIP(13); TIC(14); TZP(9); TIM(9); CTX(13); CAZ(11); FEP(8); ATM(8); IPM(4); MEM(7); ETP(0); DOR(0); CIP(7); AN(13); NET(9); TOB(8); LVX(4); CL(8) GM(7); PIP(8); TIC(7); TZP(6); TIM(7); CTX(6); CAZ(6); FEP(5); ATM(3); IPM(7); MEM(3); ETP(0); DOR(0); CIP(5); AN(7); NET(8); TOB(3); LVX(2); CL(3)
C. freundii n = 6 E. sakazaki n = 8 E. cloacae n = 19 P. aeruginosa n = 17 P. mirabilis n = 11
n = number of tested strains; values in the brackets represent the number of resistant strains.
demonstrated by the isolates to antibiotics was not surprising, as part of the inclusion criteria for the study was the presence of a difficult-to-treat infection. However, the great sensitivity of these Gram-negative pathogens to low concentrations of geranium oil is more interesting. Previous studies have mainly described the activity of geranium oil against S. aureus strains. Edwards-Jones at al. demonstrate the antibacterial properties of geranium oil against MRSA strains, including those derived from the wounds of burn patients [9]. Our own previous studies confirm the high effectiveness of oil against clinical strains of S. aureus from several types of clinical materials [18]. S. aureus strains isolated from skin lesions were found to be sensitive to geranium oil at concentrations from 0.25 ml/ml to 1.5 ml/ml, and those from postoperative wounds at concentrations ranging from 0.5 ml/ml to 2.25 ml/ml. The largest number of MRSA and MSSA clinical strains, as well as those with the MLSB mechanism, were inhibited at a 1.0 ml/ml concentration of geranium oil. Relatively few studies have addressed the susceptibility of Gram-negative bacteria to geranium oil. Probuseenivasan et al. evaluated the activity of geranium oil against standard strains by using disk diffusion and the agar dilution methods [19]. The geranium essential oil was found to inhibit the growth of Gram-positive strains such as S. aureus ATCC 25923 and Bacillus subtilis MTCC 441, as well as Gram-negative strains such as E. coli ATCC 25922, K. pneumoniae ATCC 15380, P. aeruginosa ATCC 27853 and Proteus vulgaris MTCC 1771. The
swabs: the Minimal Inhibitory Concentration was from 3.0 ml/ ml to 3.75 ml/ml. Higher MIC values, between 5.25 and 5.75 ml/ ml were obtained against the isolated strains of C. freundii. Concentrations from 6.25 ml/ml to 8.0 ml/ml inhibited the growth of all Enterobacter strains. Geranium essential oil at concentrations of 6.25–7.0 ml/ml inhibited the growth of E. sakazakii, E. cloacae were inhibited by concentrations of 7.0– 8.0 ml/ml. The least sensitive to geranium oil were strains of Pseudomonas and Proteus genera, the MIC values for both genera were from 9.25 ml/ml to 10.5 ml/ml. No correlation was found between the antibiotic resistance of the bacterial strains and their sensitivity to geranium essential oil. The susceptibility to geranium essential oil of the isolated strains is presented in Fig. 1.
5.
Discussion
In this study, it was shown that geranium oil exhibits strong antibacterial activity against Gram-negative pathogens responsible for difficult-to-treat wound infections. Geranium oil containing mainly citronellol (26.7%) and geraniol (13.4%) inhibited the growth of all Gram-negative clinical strains of E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa at concentrations from 3.0 ml/ml to 10.5 ml/ml. Susceptibility testing showed that the Gram-negative pathogens isolated from patients with wound infections are characterized by very high drug resistance. The resistance 10 9
number of streins
8 7 6 5 4 3 2 1 0 3.00
3.25
3.50
3.75
5.25
5.50
5.75
6.25
6.75
7.00
7.25
7.50
7.75
8.00
9.25
10.00
10.25
10.50
oil concentraon [ l/ml] Escherichia coli
Citrobacter freundii
Enterobacter sakazakii
Enterobacter cloacae
Pseudomonas aeruginosa
Proteus mirabilis
Fig. 1 – The susceptibility of the clinical strains isolated from wound swabs to geranium essential oil. Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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Minimal Inhibitory Concentration for geranium oil was found to be >6.4 mg/ml against E. coli, and >12.8 mg/ml against other Gram-negative reference strains, and it was seen to be active at a concentration of 1:5 against K. pneumoniae (inhibition zone of 9.0 mm) and E. coli (inhibition zone of 10.4 mm). P. aeruginosa was found to be susceptible to geranium oil at a concentration of 1:10 (inhibition zone of 9.4 mm) and P. vulgaris at 1:20 (inhibition zone of 8.3 mm). Our research work on clinical bacterial strains from wound infections has yielded similar results. The strains which were most sensitive to geranium oil were E. coli, and P. aeruginosa is more sensitive than Proteus vulgaris. The Minimal Inhibitory Concentration for E. coli clinical isolates was from 3.0 ml/ml to 3.75 ml/ml. For Pseudomonas and Proteus, MIC values were from 9.25 ml/ml to 10.5 ml/ml and 9.25 ml/ml to 10.25 ml/ml, respectively. Essential oils, thanks to their antimicrobial and antiinflammatory properties, have aroused much interest in the treatment of many dangerous infections caused by resistant pathogens. One of the best-known examples of the successful use of essential oils in treatment is the essential oil from Melaleuca alternifolia, tea tree oil. Edmondson et al. confirm that tea tree oil can be used as an effective decolonisation agent against MRSA from acute and chronic wounds of mixed etiology [20]. After treatment with a 3% solution of essential oil applied directly on the affected site, no MRSA was detected in wound swabs and the number of wounds were reduced. Halco´n and Milkus describe Melaleuca alternifolia oil as a promising adjunctive wound treatment [21]. Its comprehensive therapeutic properties are also closely linked to synergistic activity. According to Bearden et al., a combination of benzethonium chloride with tea tree oil and white thyme oil demonstrated greater activity than polymyxin B with gramicidin or neomycin with polymyxin B, as found in topical wound care products, against methicillin-resistant S. aureus [22]. Karpanen et al. demonstrate thymol, tea tree oil and eucalyptus oil to have high antimicrobial efficacy against planktonic and biofilm cultures of S. epidermidis, and note that a combination of chlorhexidine digluconate and eucalyptus oil has synergistic activity against S. epidermidis biofilm [23]. According to Riella et al., thyme essential oil significantly reduces edema and diminishes the influx of leukocytes to the injured area during wound infection. Wounds dressed with COLTHY films show greater wound retraction rates and an improved granulation reaction [24]. Roasto et al. demonstrate that geranium oil reduces the effective Minimum Inhibitory Concentration of norfloxacin when used in combination against standard strains of S. aureus [25]. Essential oils are an interesting area of study not only because of their antibacterial properties, but also their antiinflammatory and immunostimulatory effects [11]. Studies concerning the activity of essential oil constituents have shown that citronellal, geranial and geraniol encourage healing in wounds in which microbial infection interferes with elastase [26]. Essential oil from Rosmarinus officinalis L. has been shown to have great potential in reducing inflammation and enhancing wound contraction, re-epithelialization, regeneration of granulation tissue, angiogenesis and collagen deposition in the wound healing process in
5
diabetic patients [27]. Day et al. draw attention to the problem of antibiotic resistance among the strains responsible for persistent infections of wounds, and underline the importance of essential oils as effective antimicrobial agents [28]. An important factor in wound infection is not only the presence of Gram-positive bacteria, but also Gram-negative bacteria such as the E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa strains isolated in this study. Many studies note that the problem of antibiotic resistance among Gram-negative bacteria is prevalent among the genera isolated in this study [29–33]. Our study of Gram-negative bacteria emphasizes the potential use of geranium oil as an active ingredient in a range of dressings for wounds after burns and ulcers.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgements The research reported in this manuscript was supported by Grant no 503/5015-02/503-01. The authors wish to thank Prof. Danuta Kalemba from Institute of General Food Chemistry, Lodz University of Technology, Poland for geranium oil analysis.
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Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds Monika Sienkiewicz a,*, Katarzyna Poznan´ska-Kurowska b,1, Andrzej Kaszuba b,1, Edward Kowalczyk c,2 a
Medical and Sanitary Microbiology Department, Medical University of Lodz, Lodz, Pl Hallera 1, 90-647, Poland Department of Dermatology, Pediatric Dermatology and Oncology, Medical University of Lodz, Kniaziewicza 1/5, 91347 Lodz, Poland c Pharmacology and Toxicology Department, Medical University of Lodz, Lodz, Pl. Hallera 1, 90-647, Poland b
article info
abstract
Article history:
Hard-to-heal wounds represent a significant problem to patients, health care professionals,
Accepted 7 November 2013
and health care system. They can be formed as a result of mechanical injuries and burns, and any co-existing chronic disease increases the risk of their emergence. Diabetics are at a
Keywords:
greater risk of developing chronic wounds because of poor circulation, slow healing times,
Geranium oil
vascular disease and neuropathy.
Inhibiting activity
The aim of this study was to determine the antimicrobial activity of geranium oil against Gram-negative bacterial clinical strains. Clinical strains were isolated from patients with
Wounds Gram-negative pathogens
difficult-to-treat wounds and a comprehensive evaluation of their sensitivity to antibiotics was carried out. The constituents of geranium oil were specified by GC–FID–MS analysis. The micro-dilution broth method was used to check the inhibition of microbial growth at various concentrations of geranium oil. The tested geranium oil was efficacious against Gramnegative pathogens responsible for problems with wound treatment. The results suggest that geranium oil may be considered an effective component of therapy in the case of frequent recurrences of infections caused by resistant pathogens. # 2013 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Hard-to-heal wounds and ulcers present serious health complications and are significant factors in the cost of hospital treatment. This problem is caused by the spread of resistance to antibiotics commonly used in clinical practice. Both Gram-positive bacteria such as Staphylococcus aureus, coagulase-negative staphylococci, beta-hemolytic Streptococcus
(S. pyogenes, S. agalactiae), and Gram-negative bacteria such as Escherichia coli, Enterobacter species, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter baumanii are pathogens of great importance in wound infections. Aerobic gram-negative rods also infect wounds late in the course of chronic wound degeneration. Pseudomonas, Acinetobacter, Stenotrophomonas are usually acquired from exogenous sources such as bathwater [1]. From the clinical point of view, significant resistant strains of staphylococci include Methicillin-Resistant
* Corresponding author. Tel.: +48 42 639 33 42; fax: +48 42 639 33 42. E-mail addresses: [email protected] (M. Sienkiewicz), [email protected] (K. Poznan´ska-Kurowska), [email protected] (E. Kowalczyk). 1
Tel.: +48 42 651 10 72; fax: +48 42 651 10 72. Tel.: +48 42 639 33 35; fax: +48 42 639 33 35. 0305-4179/$36.00 # 2013 Elsevier Ltd and ISBI. All rights reserved. http://dx.doi.org/10.1016/j.burns.2013.11.002 2
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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S. aureus – MRSA, S. epidermidis – MRSE and S. aureus strains with reduced susceptibility to vancomycin and strains of S. aureus resistant to vancomycin [2]. The presence of constitutive and inductive Macrolide–Lincosamide–Streptogramin B – MLSB resistance mechanisms in streptococci limits the therapeutic possibilities. M-phenotype related to mef(A) gene conditioning resistance to erythromycin and other macrolides [3]. ExtendedSpectrum Beta-Lactamases (ESBLs) – enzymes produced by Gram-negative pathogens as Escherichia, Enterobacter, Klebsiella hydrolyse all penicillin-based antibiotics, monobactams and cephalosporins, even those with a wide range of activity. Multiresistant P. aeruginosa and Acinetobacter are resistant to fluoroquinolones, extended-spectrum cephalosporins, carbapenems and aminoglycosides. Clinical strains of Pseudomonas and Proteus produce metallo-beta-lactamase enzymes (Znblactamase) responsible for resistance to carbapenems [4–6]. Many kinds of dressings are used in wound treatment: absorbent, nonadherent, occlusive and semiocclusive, hydrophilic and hydrophobic, hydrocolloid and hydrogel, as well as alginates and absorbable materials, many of which could be a carrier for the testing of new, more effective antimicrobial drugs. The increasing presence of antibiotic-resistant pathogens strains has aroused interest in topical antimicrobial agents. Wound dressings containing silver have broad spectrum antimicrobial activity that encompasses many multidrug resistant wound pathogens. Jones et al. report that dressings with silver have great antibacterial effectiveness against methicillin-resistant S. aureus (MRSA) and P. aeruginosa [7,8]. Edwards-Jones et al. demonstrate that geranium essential oil vapors have strong antibacterial effects when used alone, against methicillin-resistant S. aureus when used in combination with grapefruit seed extract, and against methicillin-sensitive S. aureus when used with tea tree oil [9]. Essential oils can also be of huge relevance in Dermatology, especially in the healing of wounds and burns thanks to their antibacterial, antiviral and antifungal activities. In addition, most have anti-inflammatory, antioxidant and anti-tumor properties [10–13]. For example, according to Maruyama et al. the cutaneous application of several essential oils, especially geranium oil, may suppress the inflammatory symptoms related to neutrophil accumulation and edema [14]. The aim of our study was to determine the antibacterial activity of geranium oil against five genera of Gram-negative clinical isolates from patients with difficult-to-treat wound infections.
2.
Materials and methods
2.1.
Bacterial strains and their identification
The samples of Gram-negative clinical strains were isolated from the swabs of patients in the Clinic of Dermatology, Pediatric Dermatology and Oncology, Medical University of Lodz, Poland in 2012. The study included 63 patients, 38 males and 25 females, with a mean age of 46–58 years, All had wounds arising during the course of diabetes or non-healing wounds after burns. Gram-negative clinical strains isolated from wounds were cultured according to standard microbiological methods with use of Columbia Agar (bioMerieux) with
5% blood and Mac Conkey Agar (bioMerieux). They were identified to the species with use of API 20 E and API 20 NE tests (bioMerieux) according to manufacturer’s instructions. The bacteria were incubated at 37 8C for 24 h.
2.2.
Preparation and GC/FID/MS analysis of essential oils
A commercial essential oil from Pelargonium graveolens Ait. was purchased from the manufacturer (POLLENA-AROMA Poland) and analyzed by GC–FID–MS in the Institute of General Food Chemistry, Lodz University of Technology, using a Trace GC Ultra apparatus (Thermo Electron Corporation) with FID and MS DSQ II detectors and FIDMS splitter (SGE). Operating conditions: apolar capillary column Rtx-1ms (Restek), 60 m 0.25 mm i.d., film thickness 0.25 mm; temperature program, 50–300 8C at 4 8C/min; SSL injector temperature 280 8C; FID temperature 300 8C; split ratio 1:20; carrier gas helium at a regular pressure 200 kPa; FID temperature 260 8C; carrier gas, helium; 0.5 mL/min; split ratio 1:20. Mass spectra were acquired over the mass range 30–400 Da, ionization voltage 70 eV; ion source temperature 200 8C. The components were identified by comparing their mass spectra with those from a laboratory-made MS library, commercial libraries (NIST 98.1, Wiley Registry of Mass Spectral Data, 8th Ed. and MassFinder 4) and with literature data [15,16] along with the retention indices on an apolar column (Rtx-1, MassFinder 4) associated with a series of alkanes with linear interpolation (C8–C26).
3.
Antimicrobial activity testing
3.1.
Susceptibility testing
The tested strains were cultivated on Columbia Agar medium and incubated at 37 8C for 24 h. Bacterial suspensions with an optical density of 0.5MF were prepared with the bioMerieux densitometer, spread on Mueller-Hinton II Agar and incubated at 37 8C for 18 h. Susceptibility testing was carried out with the use of the disk-diffusion method, the following antibiotics (Becton Dickinson) being used against all tested E. coli, Citrobacter freundii, Enterobacter sakazakii, Enterobacter cloacae, P. aeruginosa and P. mirabilis clinical strains: GM – gentamicin (10 mg), PIP – piperacillin (100 mg), TIC – ticarcillin (75 mg), TZP – piperacillin/tazobaktam (100/10 mg), TIM – ticarcillin/clavulanic acid (75 mg/10 mg), CTX – cefotaxim (30 mg), CAZ – ceftazidime (30 mg), FEP–cefepim (30 mg), ATM–aztreonam (30 mg), IPM – imipenem (10 mg), MEM – meropenem (10 mg), ETP – ertapenem (10 mg), DOR – doripenem (10 mg), CIP – ciprofloxacin (5 mg), AN – amikacin (30 mg), NET – netilmicin (30 mg), TOB – tobramycin (10 mg). For clinical strains of E. coli, C. freundii, E. sakazakii and E. cloacae: CXM – cefuroxime (30 mg), C – chloramphenicol (30 mg), TE – tetracycline (30 mg), TGC – tigecyclin (15 mg), SXT – trimethoprim/sulfamethoxazole (1.25 mg/23.75 mg), only for E. coli strains: AMC – amoxicillin/ clavulanic acid (20 mg/10 mg), CF – cephalothin (30 mg), CZ – cefazolin (30 mg), AM – ampicillin (10 mg), FOX – cefoxitin (30 mg) and only for P. aeruginosa and P. mirabilis strains: LVX – levofloxacin (5 mg), CL – colistin (50 mg) additionally being used.
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [17].
3.2.
Minimal inhibitory concentration (MIC) assay
The antibacterial activity of the tested oil was investigated by the micro-dilution broth method. The essential oil was diluted in ethanol. This stock solution was mixed with a 100 ml Mueller-Hinton broth to obtain concentrations from 2.0 ml/ml to 9.0 ml/ml. An inoculum containing 1.5 108 CFU (10 ml) per well was added to broth with various oil concentrations; one well was filled with broth without oil to act as a control for strain growth. The mixtures were then transferred to 96-well microtiter plates. The Minimal Inhibitory Concentration (MIC) was determined as the lowest concentration of oil which inhibits the visible growth of bacteria after 24 h of incubation at 37 8C under aerobic conditions. The control media containing only alcohol at concentrations used in the dilutions of geranium oil did not inhibit the growth of bacterial strains.
4.
Results
4.1.
Tested Gram-negative bacterial strains
Sixty-three samples of wound swabs from patients were examined in the present study. Seventy five clinical strains of bacteria were isolated from swabs, among them were E. coli (n = 14, 18.6%), C. freundii (n = 6, 8.0%), E. sakazakii (n = 8, 10.6%), E. cloacae (n = 19, 25.3%), P. mirabilis (n = 11, 14.6%) and P. aeruginosa (n = 17, 22.6%).
4.2.
Constituents of the tested geranium oil
The results of the tested geranium oil analysis revealed that the main components were citronellol (26.7%) and geraniol (13.4%). Among sixty-one constituents identified in the geranium oil, the prevalent compounds included nerol (8.7%), citronellyl formate (7.1%), isomenthone (6.3%), linalool (5.2%), 10-epi-geudesmol (4.4%), geranyl formate (2.5%), menthone (1.6%), bcaryophyllene (1.5%), geranyl butyrate (1.4%), cis-rose oxide (1.4%), geranial (1.1%), and b-baurobonene (1.1%). The constituents of the tested oil are shown in Table 1.
4.3.
Bacterial susceptibility testing to antibiotics
The number of resistant strains to recommended antibiotics of the seventy-five clinical strains of E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa are shown in Table 2. The results show that the strains isolated from difficult-to-treat wound infections in the study were highly resistant to most of the b-lactam, cephalosporin, aminoglycoside and quinolone antibiotics recommended for susceptibility testing. Most strains were resistant to b-lactam antibiotics with inhibitors: amoxicillin with clavulanic acid, piperacyllin with tazobaktam and tikarcillin with clavulanic acid, which were used according to Clinical and Laboratory Standards Institute recommendations. Some of the E. coli, E. cloacae, P. mirabilis and P. aeruginosa strains isolated were resistant to
Table 1 – The main constituents of geranium essential oil. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Compound
%
RI
a-Pinene Limonene cis-Linlool oxide (f) Linalool cis-Rose oxide trans-Rose oxide Menthone Isomenthone a-Terpineol Citronellol Nerol Geraniol Geranial Citronellyl formate Geranyl formate Bicycloelemene Citronellyl acetate a-Cubebene Geranyl acetate a-Copaene b-Bourbonene 1,5-di-epi-Bourbonene b-Caryophyllene Citronellyl propionate b-Copaene Guaia-6,9-diene 4bH,10aH-Guaia-1(5),6-diene Geranyl propionate Alloaromadendrene 7aH,10bH-Cadina-1(6),4-diene Germacrene D b-Selinene Bicyclogermacrene a-Muurolene g-Cadinene trans-Calamenene d-Cadinene Zonarene Selina-4(15),7(11)-diene Geranyl butyrate Phenylethyl tiglate 10-epi-g-Eudesmol Geranyl tiglate Geranyl ester I
0.7 0.2 0.3 5.2 1.4 0.6 1.6 6.3 0.3 26.7 8.7 13.4 1.1 7.1 2.5 0.4
929 1021 1058 1086 1097 1113 1133 1144 1173 1217 1220 1243 1246 1261 1283 1334
0.2 0.4 0.5 1.1 0.2 1.5 0.3 0.2 0.3 0.5 1.0 0.2 0.2 1.0 0.2 0.7 0.2 0.6 0.3 0.9 0.2 0.2 1.4 0.7 4.4 1.0 0.2
1349 1361 1377 1385 1388 1419 1425 1428 1439 1445 1452 1459 1469 1477 1482 1491 1496 1509 1510 1515 1518 1530 1537 1554 1613 1675 1694
% Percentage of constituents; RI, Retention Index. The content of eighteen remaining constituents: Myrcene, a-Phellandrene, pCymene, (E)-b-Ocimene, trans-Linlool oxide (f), Isopulegol, Isomenthol, Estragole, Neryl formate, a-Gurjunene, 4aH,10aH-Guaia1(5),6-diene, g-Muurolene, g-Selinene, Dihydroagarofuran, Cadina1,4-diene, Geranyl isovalerate, g-Eudesmol, and Geranyl ester II amounted to 0.1%.
monobactam – aztreoname. The tested strains were generally sensitive to carbapenems, but few of E. coli strains were resistant to meropenem. Most of P. aeruginosa and P. mirabilis clinical isolates were resistant to carbapenems: meropenem and imipenem.
4.4.
Gram-negative bacteria susceptibility to geranium oil
Geranium essential oil demonstrated the greatest antibacterial activity against E. coli clinical strains isolated from wound
Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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Table 2 – Antibiotic resistance demonstrated by Gram-negative clinical bacterial strains isolated from wound swabs. Tested Gram-negative bacteria E. coli n = 14
Resistance to tested antibiotics
GM(10); PIP(10); TIC(11); TZP(6); TIM(9), CTX(8); CAZ(7); FEP(5); ATM(3); IPM(0); MEM(3); ETP(0); DOR(0); CIP(3); AN(7); NET(8); TOB(2); CXM(2); C(10); TE(13); TGC(3); SXT(8); AmC(11); CF(4); CZ(4); AM(14); FOX(8); GM(3); PIP(3); TIC(3); TZP(4); TIM(3); CTX(4); CAZ(0); FEP(0); ATM(0); IPM(0); MEM(0); ETP(0); DOR(0); CIP(2); AN(4); NET(2); TOB(5); CXM(6); C(6); TE(6); TGC(0); SXT(3) GM(7); PIP(0); TIC(8); TZP(4); TIM(8); CTX(0); CAZ(0); FEP(0); ATM(0); IPM(0); MEM(0); ETP(0); DOR(0); CIP(4); AN(8); NET(8); TOB(4); CXM(7); C(6); TE(4); TGC(2); SXT(8) GM(19); PIP(4); TIC(15); TZP(3); TIM(12); CTX(6); CAZ(4); FEP(3); ATM(4); IPM(0); MEM(0); ETP(0); DOR(0); CIP(5); AN(9); NET(16); TOB(10); CXM(14); C(16); TE(7); TGC(5); SXT(15) GM(12); PIP(13); TIC(14); TZP(9); TIM(9); CTX(13); CAZ(11); FEP(8); ATM(8); IPM(4); MEM(7); ETP(0); DOR(0); CIP(7); AN(13); NET(9); TOB(8); LVX(4); CL(8) GM(7); PIP(8); TIC(7); TZP(6); TIM(7); CTX(6); CAZ(6); FEP(5); ATM(3); IPM(7); MEM(3); ETP(0); DOR(0); CIP(5); AN(7); NET(8); TOB(3); LVX(2); CL(3)
C. freundii n = 6 E. sakazaki n = 8 E. cloacae n = 19 P. aeruginosa n = 17 P. mirabilis n = 11
n = number of tested strains; values in the brackets represent the number of resistant strains.
demonstrated by the isolates to antibiotics was not surprising, as part of the inclusion criteria for the study was the presence of a difficult-to-treat infection. However, the great sensitivity of these Gram-negative pathogens to low concentrations of geranium oil is more interesting. Previous studies have mainly described the activity of geranium oil against S. aureus strains. Edwards-Jones at al. demonstrate the antibacterial properties of geranium oil against MRSA strains, including those derived from the wounds of burn patients [9]. Our own previous studies confirm the high effectiveness of oil against clinical strains of S. aureus from several types of clinical materials [18]. S. aureus strains isolated from skin lesions were found to be sensitive to geranium oil at concentrations from 0.25 ml/ml to 1.5 ml/ml, and those from postoperative wounds at concentrations ranging from 0.5 ml/ml to 2.25 ml/ml. The largest number of MRSA and MSSA clinical strains, as well as those with the MLSB mechanism, were inhibited at a 1.0 ml/ml concentration of geranium oil. Relatively few studies have addressed the susceptibility of Gram-negative bacteria to geranium oil. Probuseenivasan et al. evaluated the activity of geranium oil against standard strains by using disk diffusion and the agar dilution methods [19]. The geranium essential oil was found to inhibit the growth of Gram-positive strains such as S. aureus ATCC 25923 and Bacillus subtilis MTCC 441, as well as Gram-negative strains such as E. coli ATCC 25922, K. pneumoniae ATCC 15380, P. aeruginosa ATCC 27853 and Proteus vulgaris MTCC 1771. The
swabs: the Minimal Inhibitory Concentration was from 3.0 ml/ ml to 3.75 ml/ml. Higher MIC values, between 5.25 and 5.75 ml/ ml were obtained against the isolated strains of C. freundii. Concentrations from 6.25 ml/ml to 8.0 ml/ml inhibited the growth of all Enterobacter strains. Geranium essential oil at concentrations of 6.25–7.0 ml/ml inhibited the growth of E. sakazakii, E. cloacae were inhibited by concentrations of 7.0– 8.0 ml/ml. The least sensitive to geranium oil were strains of Pseudomonas and Proteus genera, the MIC values for both genera were from 9.25 ml/ml to 10.5 ml/ml. No correlation was found between the antibiotic resistance of the bacterial strains and their sensitivity to geranium essential oil. The susceptibility to geranium essential oil of the isolated strains is presented in Fig. 1.
5.
Discussion
In this study, it was shown that geranium oil exhibits strong antibacterial activity against Gram-negative pathogens responsible for difficult-to-treat wound infections. Geranium oil containing mainly citronellol (26.7%) and geraniol (13.4%) inhibited the growth of all Gram-negative clinical strains of E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa at concentrations from 3.0 ml/ml to 10.5 ml/ml. Susceptibility testing showed that the Gram-negative pathogens isolated from patients with wound infections are characterized by very high drug resistance. The resistance 10 9
number of streins
8 7 6 5 4 3 2 1 0 3.00
3.25
3.50
3.75
5.25
5.50
5.75
6.25
6.75
7.00
7.25
7.50
7.75
8.00
9.25
10.00
10.25
10.50
oil concentraon [ l/ml] Escherichia coli
Citrobacter freundii
Enterobacter sakazakii
Enterobacter cloacae
Pseudomonas aeruginosa
Proteus mirabilis
Fig. 1 – The susceptibility of the clinical strains isolated from wound swabs to geranium essential oil. Please cite this article in press as: Sienkiewicz M, et al. The antibacterial activity of geranium oil against Gram-negative bacteria isolated from difficult-to-heal wounds. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.11.002
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Minimal Inhibitory Concentration for geranium oil was found to be >6.4 mg/ml against E. coli, and >12.8 mg/ml against other Gram-negative reference strains, and it was seen to be active at a concentration of 1:5 against K. pneumoniae (inhibition zone of 9.0 mm) and E. coli (inhibition zone of 10.4 mm). P. aeruginosa was found to be susceptible to geranium oil at a concentration of 1:10 (inhibition zone of 9.4 mm) and P. vulgaris at 1:20 (inhibition zone of 8.3 mm). Our research work on clinical bacterial strains from wound infections has yielded similar results. The strains which were most sensitive to geranium oil were E. coli, and P. aeruginosa is more sensitive than Proteus vulgaris. The Minimal Inhibitory Concentration for E. coli clinical isolates was from 3.0 ml/ml to 3.75 ml/ml. For Pseudomonas and Proteus, MIC values were from 9.25 ml/ml to 10.5 ml/ml and 9.25 ml/ml to 10.25 ml/ml, respectively. Essential oils, thanks to their antimicrobial and antiinflammatory properties, have aroused much interest in the treatment of many dangerous infections caused by resistant pathogens. One of the best-known examples of the successful use of essential oils in treatment is the essential oil from Melaleuca alternifolia, tea tree oil. Edmondson et al. confirm that tea tree oil can be used as an effective decolonisation agent against MRSA from acute and chronic wounds of mixed etiology [20]. After treatment with a 3% solution of essential oil applied directly on the affected site, no MRSA was detected in wound swabs and the number of wounds were reduced. Halco´n and Milkus describe Melaleuca alternifolia oil as a promising adjunctive wound treatment [21]. Its comprehensive therapeutic properties are also closely linked to synergistic activity. According to Bearden et al., a combination of benzethonium chloride with tea tree oil and white thyme oil demonstrated greater activity than polymyxin B with gramicidin or neomycin with polymyxin B, as found in topical wound care products, against methicillin-resistant S. aureus [22]. Karpanen et al. demonstrate thymol, tea tree oil and eucalyptus oil to have high antimicrobial efficacy against planktonic and biofilm cultures of S. epidermidis, and note that a combination of chlorhexidine digluconate and eucalyptus oil has synergistic activity against S. epidermidis biofilm [23]. According to Riella et al., thyme essential oil significantly reduces edema and diminishes the influx of leukocytes to the injured area during wound infection. Wounds dressed with COLTHY films show greater wound retraction rates and an improved granulation reaction [24]. Roasto et al. demonstrate that geranium oil reduces the effective Minimum Inhibitory Concentration of norfloxacin when used in combination against standard strains of S. aureus [25]. Essential oils are an interesting area of study not only because of their antibacterial properties, but also their antiinflammatory and immunostimulatory effects [11]. Studies concerning the activity of essential oil constituents have shown that citronellal, geranial and geraniol encourage healing in wounds in which microbial infection interferes with elastase [26]. Essential oil from Rosmarinus officinalis L. has been shown to have great potential in reducing inflammation and enhancing wound contraction, re-epithelialization, regeneration of granulation tissue, angiogenesis and collagen deposition in the wound healing process in
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diabetic patients [27]. Day et al. draw attention to the problem of antibiotic resistance among the strains responsible for persistent infections of wounds, and underline the importance of essential oils as effective antimicrobial agents [28]. An important factor in wound infection is not only the presence of Gram-positive bacteria, but also Gram-negative bacteria such as the E. coli, C. freundii, E. sakazakii, E. cloacae, P. mirabilis and P. aeruginosa strains isolated in this study. Many studies note that the problem of antibiotic resistance among Gram-negative bacteria is prevalent among the genera isolated in this study [29–33]. Our study of Gram-negative bacteria emphasizes the potential use of geranium oil as an active ingredient in a range of dressings for wounds after burns and ulcers.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgements The research reported in this manuscript was supported by Grant no 503/5015-02/503-01. The authors wish to thank Prof. Danuta Kalemba from Institute of General Food Chemistry, Lodz University of Technology, Poland for geranium oil analysis.
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