Antibacterial effect of oregano essential oil alone and in combination with antibiotics against extended-spectrum β-lactamase-producing Escherichia coli
Correspondence: Zhen-ling Zeng, Guangdong Provincial Key Laboratory of Veterinary, Pharmaceutics Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Road, Guangzhou, Guangdong 510642, China. Tel.: +86 20 8528 0665; fax: +86 20 8528 4896; e-mail: email@example.com
In this paper, we studied the antibacterial effects of oregano essential oil (OEO) both alone, using a twofold dilution method, and combined with antibiotics, using a checkerboard microtitre assay, against extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. The result indicated that multiple drug-resistant E. coli was very sensitive to OEO and polymycin; their minimal inhibitory concentration values are 0.5 μL mL−1 and 0.8 μg mL−1. The antibacterial effects of OEO in combination with kanamycin were independent against E. coli, with fractional inhibitory concentration (FIC) indices of 1.5. The antibacterial effects of OEO combined with amoxicillin, polymycin, and lincomycin showed an additive effect against E. coli, with FIC indices in the range of 0.625–0.750. The antibacterial effects of OEO in combination with fluoroquinolones, doxycycline, lincomycin, and maquindox florfenicol displayed synergism against E. coli, with FIC indices ranging from 0.375 to 0.500. The combination of OEO with fluoroquinolones, doxycycline, lincomycin, and maquindox florfenicol to treat infections caused by ESBL-producing E. coli may lower, to a great extent, the effective dose of these antibiotics and thus minimize the side effects of antibiotics. This is the first report on OEO against ESBL-producing E. coli.
With the misuse and overuse of antibiotics to treat diseases, resistance to the drugs has begun to appear and has become more serious because of selective pressure. Resistance to multiple drugs is common for some kinds of clinically isolated bacterial strains; one of the mechanisms in multiple drug resistance is that bacteria can produce extended-spectrum β-lactamases (ESBLs) (Jacoby & Medeiros, 1991; Perilli et al., 1997). ESBL-producing Enterobacteriaceae and other kinds of bacteria have been reported widely. Infections caused by ESBL-producing bacteria have become a clinical and therapeutic problem because these organisms are resistant not only to β-lactamases but also to many other antimicrobial agents (Velasco et al., 2007). Often, ESBL-producing bacterial strains were isolated from animals in the veterinary field as well. It is important to prevent bacteria from producing ESBLs and to control diseases caused by ESBL-producing pathogens. The best way of treating these diseases is with imipenem and meropenem and third-generation cephalosporins in combination with enzyme inhibitors; however, these drugs cannot be used extensively in the veterinary field because of their high prices. Therefore, more economical and effective antibacterial agents are urgently needed in the veterinary field.
A feasible approach to limiting the transmission of these pathogens is to use essential oils as alternative agents or topical agents. Oregano, one kind of labiate Origanum plant that has been known for a long time as a popular remedy, is a very versatile plant. It was reported that Origanum compactum, Origanum minutif lorum, and Origanum majorana exhibit antifungal activity, antibacterial activity, and antimicrobial activity, respectively (Bouchra et al., 2003; Baydar et al., 2004; Vági et al., 2005). Until now its potential therapeutic roles such as diaphoretic, carminative, antispasmodic, antiseptic, and tonic properties have been recognized (Nostro et al., 2004). Sometimes OEO in conjunction with VP/MAP at chill temperatures can be used as a means of controlling the spoilage and safety of meat (Skandamis et al., 2002). In addition, it has been used widely in China as a kind of feed additive because it has a broad spectrum of action against bacteria, a rapid effect, and little residue, and now its antibacterial effect has been researched in vitro (Sivropoulou et al., 1996; Dorman & Deans, 2000; Force et al., 2000; Aligiannis et al., 2001; Lambert et al., 2001; Manohar et al., 2001) and in vivo (Adam et al., 1998) throughout the world. There is a lack of knowledge about OEO, which is one kind of volatile oil of Origanum vulgare linn (O. vulgare L.), used against ESBL-producing E. coli isolated from poultry in the veterinary field, especially on its combination with other antibacterial agents. The mechanism may be in its principal components, i.e. thymol and carvacrol, which have antimicrobial properties.
The objective of the present work was to investigate the antibacterial effects of OEO against ESBL-producing E. coli. The effects of OEO in combination with antibiotics were also evaluated in order to seek effective combinations to treat emergent infections caused by ESBL-producing E. coli. This is the first report on OEO alone and in combination with antibacterial agents against ESBL-producing E. coli isolated from chicken.
Materials and methods
Escherichia coli isolates were obtained from chicken livers from a chicken farm at Henan Agricultural University in Zhengzhou, Henan Province, China. Strains were isolated and purified on an agar plate and identified using a Vitek 32 system and GNI+card (bioMérieux, France).
Screening for and confirmation of ESBLs
To detect the ESBLs, the initial screening was performed by testing the zone diameter for ceftriaxone (30 μg), ceftazidime (30 μg), cefotaxim (30 μg), and aztreonam (30 μg) and interpreted by the criterion of NCCLS (Adam et al., 1998; Aligiannis et al., 2001), where a positive result was considered to be suspicious for the presence of ESBLs. This test was followed by a phenotypic confirmation test of ESBLs, carried out by testing the zone diameter for cefotaxim (30 μg) and ceftazidime (30 μg) with and without clavulanate (10 μg), as described previously by the NCCLS. A 5 mm increase in the zone diameter for ceftazidime or cefotaxim tested in combination with clavulanate vs. the zone when tested alone was considered indicative of ESBL production. Klebsiella pneumoniae American Type Culture Collection (ATCC) 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively (Adam et al., 1998; Dorman & Deans, 2000).
Identification of ESBLs gene
Templates of total DNA from the isolate were prepared as described previously and the identity of the ESBLs was determined by PCR amplification of selected determinants followed by direct sequencing of the amplicons as described previously. The primers used for detecting ESBLs−TEM genes were as follows: the forward primer (5′-GGGGATGAGTATTCAACATTTCC-3′) and the reverse primer (5′-GGGCAGTTACCAATGCTTAATCA-3′) were designed using oligo 6.0 software based on the published ESBL gene sequence (Genbank accession no. AF332513). The sequence region of an 861-bp fragment containing an ORF of a full-length ESBL gene was predicted between the two primers. PCR was carried out in a final volume of 50 μL containing 1 μg template DNA, 100 pmol of each primer (flo1/flo2), 1 × PCR buffer, 0.2 mM of each of the dNTPs, and 2.5 U Ex Taq polymerase (Takara, Japan). A total of 30 cycles were performed in the PCR Express (HYBRID Corporation), under the following conditions: denaturation at 94 °C for 1 min, annealing at 53 °C for 30 s, and extension at 72 °C for 1 min with final termination at 72 °C for 10 min.
The PCR products were electrophoresed on 1.0% agarose gel. The DNA band of interest was excised, purified, and ligated to pGEM-T Easy vector (Promega), and the recombinant vector named pGEM-ESBL was constructed. JM109 competent cells were transformed, and the white clones were screened and digested with restriction enzyme EcoRI and sequenced for identification.
Oregano essential oil (OEO)
OEO was collected as previously described (Nostro et al., 2004). The aerial parts of oregano (O. vulgare L.), obtained from a commercial source (Yuanzheng Ltd, Hebei, China), were subjected to hydrodistillation for 2 h using a modified clevenger type apparatus, and the essential oil was collected and stored at 4 °C. All oils were diluted (v/v) by both the agar and broth dilution methods.
Determination of minimal inhibitory concentration (MIC)
The isolated strain was stored at −70 °C until use; E. coli was grown in Martin broth modified and adjusted with medium to match the 0.5 McFarland standard (105 CFU mL−1). The MICs of OEO, defined as the lowest concentration that completely inhibited visible bacterial growth after 14 h, and antibiotics were examined by broth dilution method in 96-well, microtitre plates. The MICs of the agents sarafloxacin, levofloxacin, polymycin, lincomycin, amoxicillin, ceftiofur, ceftriaxone, maquindox, florfenicol, doxycycline, and kanamycin against the isolated strain were determined by the twofold dilution method, and the results were interpreted in accordance with the recommendations of the National Committee for Clinical Laboratory standards (Adam et al., 1998; Dorman & Deans, 2000). Escherichia coli strain ATCC 25922 was used as quality control in MIC determination.
Organisms were measured by twofold dilutions of OEO samples with Martin broth modified solutions containing 0.5% Tween-80 to enhance oil solubility. The concentration of essential oil in the medium ranged from 0.5% to 0.0039% (v/v). All determinations were performed in triplicate. If MIC values were different, the trial was repeated until the values were the same. In this way, the result is certain. Two growth controls consisting of Martin broth modified medium and Martin broth modified with 1.0% Tween-80 were added to ascertain that these vehicles did not affect bacterial growth.
Checkerboard microtitre test
Ten serial, twofold dilutions of the bacterial fraction and essential oil were prepared using the same solvents as in the MIC tests. Aliquots (50 μL) of antibacterial dilution were added to the wells of a 96-well plate in a vertical orientation and 10 μL aliquots of OEO dilution were added in a horizontal orientation so that the plate could contain various concentration combinations of the two compounds. Then, each well was inoculated with 100 μL (c. 5 × 105 CFU well−1) of antibacterial bacterial suspensions and cultivated at 37 °C. Fractional inhibitory concentration (FIC) was calculated by dividing the MIC of the combination of antibacterial and essential oil by the MIC of antibacterial or essential oil alone. The FIC index, obtained by adding both FICs, was interpreted as synergistic when it was ≤0.5, as additive (indifferent) when it was >0.5 and ≤2.0, and as antagonistic when it was >2.0 (White et al., 1996). Similar checkerboard experiments were performed as well to test the combined effect of the essential oil with each antibacterial.
Results and discussion
The initial screening was carried out to test inhibition zones of ceftriaxone, ceftazidime, cefotaxim and aztreonam, and the corresponding inhibitory diameters were 14 mm, 16 mm, 17 mm, and 14 mm, respectively, which were consistent with the criterion of NCCLS. Thus, it was tentative that this strain might produce >ESBLs. Moreover, the confirmation test of ESBL production indicated that the inhibition zones of cefotaxim and cefotaxime-plus-clavulanate disks, ceftazidime and ceftazidime-plus-clavulanate disks were 14 mm, 20 mm, 15 mm and 17 mm, respectively. A distance of 6 mm between the zones of cefotaxim and cefotaxim-plus-clavulanate disks was considered to indicate the production of ESBLs. Besides, the resulting PCR product of the ESBLs−TEM gene was about 861 bp in length and identical to the expected size. Further identification of the restriction enzyme and sequencing of recombinant plasmid pGEM-ESBLs undoubtedly confirmed that the isolate carried the ESBLs-TEM gene. Sequence analysis of the 861-bp amplicon produced with the TEM-specific primers showed that the plasmid harbored a TEM-type gene with 100% identity to TEM-116 (AJ847364); the result revealed that the 287-amino-acid protein was the same type gene with 100% identity to TEM-116 (AJ847364). TEM-116 is one of primary, prevalent ESBL types. It is remarkable that there is 22.1% TEM-116 in a survey of ESBL-producing pathogens in a Spanish University Hospital; especially 49.32% TEM-116 pathogens can produce other ESBL types at the same time (Romero et al., 2007). More than 400 different types of β-lactamases originating from clinical isolates have been described. Since they were first identified at the beginning of the 1980s (Perilli et al., 1997), ESBL-producing Enterobacteriaceae have been reported widely (Jarlier et al., 1988; Sirot et al., 1988; Jacoby & Medeiros, 1991; Yang et al., 1998; Bonnet et al., 1999). ESBL occurred predominantly not only in Klebsiella species and E. coli but also in other genera of the family Enterobacteriaceae, such as Shigella and others (Ashkenazi et al., 2003; Ahmed et al., 2004). Organisms producing ESBLs are clinically relevant and remain an important cause for failure of therapy with cephalosporins. It is very important to find some effective drugs against these diseases.
The susceptibility test results of the drug alone suggested that ESBLs-producing E. coli had serious resistance to commonly used antibacterial agents including fluoroquinolones, doxycycline, lincomycin, maquindox, florfenicol, and amoxicillin, and most of their MIC values were over 16 μg mL−1, indicating that the ESBL-producing E. coli had serious resistance to multiple drugs. This result was consistent with some recent surveys that ESBL-producing E. coli exhibited cross-resistance to tetracycline, gentamicin, and ciprofloxacin. ESBL-producing pathogens are often encoded by genes located on large plasmids, and these also carry a gene for resistance to other antibiotics (Bradford, 2001; Rahal, 2002).
Thus, this would probably bring many difficulties in treating diseases caused by these bacteria. However, this strain was sensitive to polymycin with the MIC value of 0.8 μg mL−1, implying that polymycin could treat the infection disease caused by these drug-resistant pathogens. It should be considered that polymycin was used to treat these diseases alone because of its poor oral absorption. Viewed from the susceptibility test result, ESBL-producing bacteria were sensitive to OEO. Moreover, the bacteria were still sensitive to the same strain when the original antibacterial compound was diluted 10 000-fold, suggesting that it was worthwhile to explore an effective alternative to antibiotics to treat diseases caused by bacteria, especially ESBL-producing bacteria.
The efficacy of OEO combined with an antibacterial agent was determined by checkboard assay, and the results are listed in Table 1. The MIC value of OEO combined with many antibacterial agents decreased predominantly, suggesting that OEO was synergistic. This is the first report concerning the synergistic effects of OEO in combination with antibiotics against bacteria, especially ESBL-producing bacteria. The MIC value of OEO in combination with most of the bacterial agents against ESBL-producing E. coli remarkably decreased, with FIC indices ranging from 0.375 to 0.75 (excluding the combination with kanamycin with an FIC index of 1.5. The FIC index indicated an independence between oil and kanamycin against ESBL-producing E. coli.) Moreover, the susceptibilities of bacteria to amoxicillin, polymycin, lincomycin, and third-generation cephalosporin were enormously improved by combination with OEO. The FIC indices of OEO combined with polymycin, lincomycin, amoxicillin, and cephalosporins were 0.75, 0.75, 0.75, and 0.625, respectively, and these data showed that these combinations had an additive effect. FIC indices of OEO combined with fluoroquinolones, doxycycline, lincomycin, maquindox, and florfenicol altered between 0.375 and 0.5. Hence the FIC indices of OEO combined with these antibiotics were synergistic.
Table 1. Concentrations of OEO (μL mL−1) and of antibacterial (μg mL−1)
|Sarafloxacin||640||80|| || |
|Levofloxacin||64||16|| || |
|Polymycin||1.6||0.8|| || |
|Lincomycin||320||160|| || |
|Amoxicillin||1280||320|| || |
|Ceftiofur||640||80|| || |
|Ceftriaxone||640||80|| || |
|Maquindox||64||16|| || |
|Florfenicol||320||40|| || |
|Doxycycline||64||16|| || |
|Kanamycin||1280||1280|| || |
Although an effect of oregano on chicken lactobacilli and E. coli was studied recently (Horosováet al., 2006), little was known about the mechanism of action for OEO on different bacteria or about the specific effects of OEO constituents, e.g. carvacrol and thymol (Cox et al., 1998; Helander et al., 1998; Ultee et al., 1999; Skandamis et al., 2000; Tassou et al., 2000). According to Conner & Beuchat (1984), the antimicrobial action of essential oils might be due to the impairment of a variety of enzyme systems, including those involved in energy production and structural component synthesis. Overall, we can conclude that OEO is one of the most promising natural compounds that can be used to develop safer antibacterial agents and that its effective combination with antibacterials may be used in the future to treat diseases caused by ESBL-producing E. coli in the veterinary field instead of expensive antibacterial agents.
This work was supported by The Research Fund for the Doctoral Program of Higher Educator (RFDP, No. 30471307).