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Keywords:

  • oregano essential oil;
  • ESBLs;
  • combination

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

Bacterial strains

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

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)
DrugsSingle drugCombined use of drugsFICsResults
  1. FIC, fractional inhibitory concentration; OEO, oregano essential oil.

OEO0.50.1250.375Synergism
Sarafloxacin64080  
OEO0.50.1250.5Synergism
Levofloxacin6416  
OEO0.50.1250.75Additive effect
Polymycin1.60.8  
OEO0.50.1250.75Additive effect
Lincomycin320160  
OEO0.50.250.75Additive effect
Amoxicillin1280320  
OEO0.50.250.625Additive effect
Ceftiofur64080  
OEO0.50.250.625Additive effect
Ceftriaxone64080  
OEO0.50.1250.5Synergism
Maquindox6416  
OEO0.50.1250.375Synergism
Florfenicol32040  
OEO0.50.1250.375Synergism
Doxycycline6416  
OEO0.50.251.5Independence
Kanamycin12801280  

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.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

This work was supported by The Research Fund for the Doctoral Program of Higher Educator (RFDP, No. 30471307).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  • Adam K, Sivropoulou A, Kokkini S, Lanaras T & Arsenakis M (1998) Antifungal activity of Origanum vulgare subsp. hirtum, Mentha spicata, Lavandula angustifolia, and Salvia fruticosa essential oils against human pathogenic fungi. J Agric Food Chem 46: 17391745.
  • Ahmed AM, Nakano H & Shimamoto T (2004) The first characterization of extended-spectrum beta-lactamase-producing Salmonella in Japan. J Antimicrob Chemother 54: 283284.
  • Aligiannis N, Kalpoutzakis E, Mitaku S & Chinou BI (2001) Composition and antimicrobial activity of the essential oils of two Origanum species. J Agric Food Chem 49: 41684170.
  • Ashkenazi S, Levy I, Kazaronovski V & Samra Z (2003) Growing antimicrobial resistance of Shigella isolates. J Antimicrob Chemother 51: 427429.
  • Baydar H, Sagdic OZkan G et al. (2004) Antibacterial activity and composition of essential oils from Origanum, Thymbra and Satureja species with commercial importance in Turkey. Food Control 15: 169172.
  • Bonnet R, De Champs C, Sirot D, Labia R & Sirot J (1999) Diversity of TEM mutants in Proteus mirabilis. Antimicrob Agents Ch 43: 26712677.
  • Bouchra C, Achouri M, Idrissi Hassani LM et al. (2003) Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botry tiscinerea Pers:Fr. J Ethnopharmacol 89: 165169.
  • Bradford PA (2001) Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14: 933951.
  • Conner DE, Beuchat LR, Worthington RE & Hitchcock HL (1984) Effects of essential oils and oleoresins of plants on ethanol production, respiration and sporulation of yeasts. Int J Food Microbiol 1: 6374.
  • Cox SD, Gustafson JE, Mann CM, Markham JL, Liew YC, Hartland RP, Bell HC, Warmington JR & Wyllie SG (1998) Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli. Lett Appl Microbiol 26: 355358.
  • Dorman HJD & Deans SG (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88: 308316.
  • Force M, Sparks WS & Ronzio A (2000) Inhibition of enteric parasites by emulsified oil of oregano in vivo. Phytother Res 14: 213214.
  • Helander IK, Alakomi HL, Latva-Kala K, Mattila-Sandholm T, Pol I, Smid EJ & Von Wright A (1998) Characterization of the action of selected essential oil components on Gram negative bacteria. J Agric Chem 46: 35903595.
  • Horosová K, Bujnáková D & Kmet V (2006) Effect of oregano essential oil on chicken lactobacilli and E. coli. Folia Microbiol (Praha) 51: 278280.
  • Jacoby GA & Medeiros AA (1991) More extended-spectrum β-lactamases. Antimicrob Agents Chemother 35: 16971704.
  • Jarlier V, Nicolas MH, Fournier G & Philippon A (1988) Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis 10: 867878.
  • Lambert RJW, Skandamis PN, Coote PJ & Nychas GJE (2001) A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol 91: 453462.
  • Manohar V, Ingram C, Gray J, Talpur NA, Echard BW, Bagchi D & Preuss G (2001) Antifungal activities of origanum oil against Candida albicans. Mol Cell Biochem 228: 111117.
  • Nostro A, Blanco AR, Cannatelli MA, Enea V, Flamini G, Morelli I, Roccaro AS & Alonzo V (2004) Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol. FEMS Microbiol Lett 230: 191195.
  • Perilli M, Felici A, Franceschini N, De Santis A, Pagani L, Luzzaro F, Oratore A, Rossolini GM, Knox JR & Amicosante G (1997) Characterization of a new TEM-derived β-lactamase produced in a Serratia marcescens strain. Antimicrob Agents Chemother 41: 23742382.
  • Rahal JJ, Urban C & Segal-Maurer S (2002) Nosocomial antibiotic resistance in multiple gram-negative species: experience at one hospital with squeezing the resistance balloon at multiple sites. Clin Infect Dis 34: 499503.
  • Romero EDV, Padilla TP, Hernández AH, Grande RP, Vázquez MF, García IG, García-Rodríguez JA & Muñoz Bellido JL (2007) Prevalence of clinical isolates of Escherichia coli and Klebsiella spp. producing multiple extended-spectrum β-lactamases. Diagn Microbiol Infect Dis 59: 433437.
  • Sirot J, Chanal C, Petit A, Sirot D, Labia R & Gerbaud G (1988) Klebsiella pneumoniae and other Enterobacteriaceae producing novel plasmid-mediated β-lactamases markedly active against third-generation cephalosporins: epidemiologic studies. Rev Infect Dis 10: 850859.
  • Sivropoulou A, Papanicolau E, Nicolaou C, Kokkini S, Lanaras T & Arsenakis M (1996) Antimicrobial and cytotoxic activities of Origanum essential oils. J Agric Food Chem 44: 12021205.
  • Skandamis P, Tsigarida E & Nychas GJE (2000) Ecophysiological attributes of Salmonella typhimurium in liquid culture and within gelatin gel with or without the addition of oregano essential oil. World J Microbiol Biotechnol 16: 3135.
  • Skandamis P, Tsigarida E & Nychas G-JE (2002) The effect of oregano essential oil on survival/death of Salmonella typhimurium in meat stored at 5 °C under aerobic, VP/MAP conditions. Food Microbiol 19: 97103.
  • Tassou CC, Koutsoumanis K & Nychas GJE (2000) Inhibition of Salmonella enteritidis and Staphylococccus aureus in nutrient broth by mint essential oil. Food Res Intern 33: 273280.
  • Ultee A, Kets EPW & Smid EJ (1999) Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 65: 46064610.
  • Vági E, Simándi B, Suhajda Á & Héthelyi É (2005) Essential oil composition and antimicrobial activity of Origanum majorana L. extracts obtained with ethyl alcohol and supercritical carbon dioxide. Food Res Int 38: 5157.
  • Velasco C, Romero L, Martínez JMR, Rodríguez-Baño J & Pascual A (2007) Analysis of plasmids encoding extended-spectrum β-lactamases (ESBLs) from Escherichia coli isolated from non-hospitalised patients in Seville. Int J Antimicrob Agents 29: 8992.
  • White RL, Burgess DS, Manduru M & Bosso JA (1996) Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother 40: 19141918.
  • Yang Y, Bhachech N, Bradford PA, Jett BD, Sahm DF & Bush K (1998) Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli isolates producing TEM-10 and TEM-43 β-lactamases from St. Louis, Missouri. Antimicrob Agents Chemother 42: 16711676.