1. Background
For the first time, Helicobacter pylori isolated from human gastric biopsies by Warren and Marshall in 1983 (1). Marshall reported that gastrointestinal disorders caused by self- ingestion of this bacterium (2). Today, this bacterium is known as the most common human pathogen affecting half of the world's population (3). The infections caused by H. pylori in human ranged from asymptomatic infections to symptomatic clinical presentations including acute or chronic gastritis, peptic ulcer diseases, gastric lymphoma and gastric adenocarcinoma (4). Eradication therapy is suggested for symptomatic patients. There are many different regimens used to treat H. pylori infections but, antibiotic resistance is the most important problems H. pylori eradication therapy (3, 5).
More than 60000 years ago, H. pylori colonized in human stomach and coevolved with humans since new human originated from his ancestors in Africa (6). Through the time H. pylori adapted to extraordinary acidic environment of gastric mucosa. The harsh environment of its niche enforces the bacterium to acquire particular mechanisms for rapid and effective adaptation to unfavorable conditions including presence of antimicrobial agents; as a result, H. pylori varied its gene pool. These variations were caused through point mutation, insertion, deletion or DNA exchange mechanisms (7).
There are some suspected mechanisms causing resistance against antibiotic agents but point mutation is accepted as the most important mechanism of antibiotic resistance in H. pylori (5). These sort of mutations occur at a very high frequency (10-5-10-8) in H. pylori, which is much larger than many other bacteria such as Enterobacteriaceae (8). Therefore, anti-mutagenic agents could be beneficial in reducing the occurrence of resistance-conferring mutations (9). In this respect, medicinal plants could be used as the potential source.
2. Objectives
In this study, we tried to evaluate anti-mutagenic properties of some plant extracts on the occurrence of mutations conferring clarithromycin resistance in H. pylori.
3. Materials and Methods
3.1. Plant Collection and Extraction
In the present study, four herbs which are used in traditional Iranian medicine, including Teucrium polium (germander), Achillea millefolium (yarrow), Thymus vulgaris (thyme) and Mirtus communis (myrtle) were purchased from a local medicinal herb shop. They were identified and confirmed by Professor G. H. Shahidi (Department of Botany, Faculty of Agriculture, Bahonar University, Kerman, Iran). The voucher numbers are kept at the Kerman University of Medical Sciences.
The seeds of M. communis, T. vulgaris leaves, flowers of T. polium and both leaves and flowers of A. millefolium were extracted as follows. The plant parts were washed, dried and grounded with a mechanical blender. Dry powders were soaked in extra pure ethanol (Merck, Germany) with solvent to sample ratio of 5:1 (v/w). After 5 days, each suspension filtered through Whatman filter paper No 5 (Whatman, England) and was concentrated, under reduced pressure. Each concentrated extract was evaporated to leave a solid pellet. The solid extracts were stored in dark at -20°C until use. Before application the solid extracts were dissolved in dimethyl solfoxide (DMSO, Merck, Germany) to a final concentration of less than 1%.
3.2. Bacterial Isolation
Three clarithromycin susceptible H. pylori isolates (K1, K7, and K11) were used in this study (see below). These isolates were obtained during a three-month-period from May to August 2011 from biopsy samples of patients referred to endoscopy unit of University hospital (Afzalipour, Kerman, Iran) in a previous study (10). All isolates were kept in brucella broth (BBL, USA) supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany) and 30% glycerol at -70°C.
3.3. Selection of Claritromycin Susceptible Isolates
Clarithromycin susceptibility was confirmed by inoculating a suspension of each isolate adjusted to a 3 of McFarland turbidity (approximately 9.0 x 108 CFU. mL-1) on Muller-Hinton agar (Merck, Germany) plates containing 10% fetal bovine serum and 1 μg clarithromycin mL-1. Clarithromycin sensitive isolates were not able to grow at such concentration (11).
3.4. Determination of Minimum Inhibitory Concentration
Final concentrations of 62.5, 125, 250, 500 and 1000 µg.mL-1 of the plant extracts were prepared for this purpose. Plant extracts were added to Muller-Hinton agar (Merck, Germany) enriched with 10% fetal bovine serum to give the above mentioned final concentrations. Plates were spot inoculated by 2 µL of a bacterial suspension adjusted to a turbidity 3 McFarland. The same plates without extract were also used as negative control. Incubation period was 3 days under micro aerobic atmosphere at 37°C (11).
The final concentrations of 0.03, 0.06, 0.125, 0.25, 0.5 and 1 µg. mL-1 of clarithromycin were also used for determination the MIC of clarithromycin for each of these three isolates. Similar method and incubation condition were applied as above mentioned. Moreover, two concentrations (500 and 1000 µg.mL-1) of each plant extract in combination with sub-inhibitory concentration (0.06 µg mL-1) of clarithromycin in agar medium were used to evaluate the possible synergistic properties. Similar medium containing 0.06 µg. mL-1 clarithromycin without any plant extracts was also used as control.
3.5. Determination of Mutation Frequency
Mutation at 23s rRNA was introduced as the main cause of clarithromycin resistance in H. pylori. Therefore, in the present study we ignored clarithromycin. Mutation frequency was calculated using Luria Delbruck fluctuation assay by enumeration of observed mutant colonies grown from 20 parallel broth cultures in Brucella agar supplemented with 10% fetal bovine serum. Therefore, we prepared 25 independent tubes containing 1 mL Brucella broth supplemented with 10% fetal bovine serum. These tubes were inoculated with 10 µL of a preliminary culture (104-105 CFU. mL-1) of each isolate and were incubated under micro aerobic condition for 3 days at 37°C. Five tubes from the above were used for enumeration of viable counts and 1 mL of each of the remaining 20 tubes was spread on agar media containing 1 μg. mL-1 clarithromycin. After a 3-day- incubation, the colonies on the agar plates were counted. Mutation frequency was calculated from the proportion of median number of counted mutant colonies in 20 tubes divided by the total viable counts (12, 13). A 50-µL- sample from preliminary culture was plated on agar media containing 1 μg. mL-1 clarithromycin to confirm no preexisting resistant mutants.
3.6. Assessment of Mutation-Inhibiting Properties of Plant Extracts
In order to determine any anti-mutational properties of the plant extracts, mutation frequency was calculated in the presence of five sub-inhibitory concentration of plant extracts (62.5, 125, 250, 500 and 1000 µg.mL-1). The results were compared with the experimental condition in absence of plant extracts. All the experiments were performed in triplicates.
3.7. DNA Extraction
DNA extraction performed using Bioneer genomic kit (Bioneer, South Korea) and the DNA extracted from all three isolates. Furthermore, DNA extracted by same method from one resistant mutant colony related to each isolate that was picked up after mutation frequency assessment.
3.8. PCR-Sequencing
A pair of oligonucleotide primers (Table 1) obtained from the known sequences of 23s rRNA gene of a wild UA802 strain (GenBank accession No. U27270) were used to detect point mutations responsible for clarithromycin resistance. A 468 base pairs (bp) fragment corresponding to position 1811-2279 of the 23s rRNA gene was amplified in 35 cycles by a gradient thermal cycler (Biometra, Germany). PCR conditions were as follows, a primary denaturation at 94°C for 7 minutes followed by 1 minute denaturation at 94°C, 1 minute annealing at 57°C, 1 minute extension at 72°C and a final extension step for 5 minutes at 72°C as the end of amplification. The amplified fragments were sent to Macrogen Company (Gasan-dong, Geumchen-gu, Seoul, Korea) for sequencing. Point mutations were detected by comparing the sequences of the mutants (resistant colonies) with those of same fragment from 23s rRNA gene of a clarithromycin sensitive H. pylori strain UA802 in the GenBank, (accession No. U27270) (14).
Table 1. Oligonucleotide Primers
| Primers | Sequences (5´→3´) | Position |
|---|---|---|
| HP-F | TAAGGTGTGCCACAGCGAT | 1811-1830 |
| HP-R | CTAACAGAAACATCAAGGG | 2279-2260 |
4. Results
4.1. Extraction Yield
Extraction yield (% w/w) was calculated from the weight of final solid extract divided to the weight of the primary powdered plant parts. Our method have an extraction yield of 5% for M. communis, 7% T. vulgaris and A. millefolium to 9% for T. polium.
4.2. Minimal Inhibitory Concentration Values
The growth of all three isolates were inhibited at the concentration of 0.125 µg. mL-1 of clarithromycin and above. MIC of clarithromycin on mutated resistant colonies raised to more than 16 µg.mL-1. Anti-Helicobacter properties of the plant extracts were evaluated at five different concentrations (62.5, 125, 250, 500 and 1000 µg.mL-1). All of the applied extracts, except M. communis in the above mentioned concentration had shown no effect on the tested bacterial isolates. Therefore, it is expected that MIC of these extracts is more than 1000 µg.mL-1. The MIC against M. communis was 1000 µg mL-1. There was not any observed synergism between M. communis concentration less than 1000 µg.mL-1 and 0.06 µg. mL-1 of clarithromycin.
4.3. Point Mutation Conferring Resistance to Clarithromycin
DNA sequences of a 468-bp-fragment of the 23s rRNA gene from preliminary isolated bacteria (K1, K7, K11) compared with DNA sequences of clarithromycin sensitive wild UA802 strain. The results were completely identical; therefore preliminary isolated bacteria were preliminary confirmed as clarithromycin susceptible. A-to-G transition at 2143 position (A2143G) was detected as major point mutation in the sequences associated with the resistant mutant colonies incorporated to each of isolates.
4.4. Mutation Frequency of Clarithromycin Resistance
Mutation frequency was separately calculated for each isolate. Some minor differences between values were observed. The overall mean value was calculated to be 27 × 10-9.
4.5. Mutation Frequency Values Applying the Plant Extracts
Results obtained from these experiments showed that at only three concentrations (250, 500 and1000 µg mL-1), the plant extract were effective in reducing the mutation frequency compared to the results obtained at the absence of plant extracts. Data collected after application of different concentrations of plant extracts were very similar (data not showed); therefore, we choose the minimal effective concentration at 250 µg mL-1 for the rest of experiments. Our findings showed a significant reduction in the mutation frequency value after application of extracts of M. communis, T. polium and A. millefolium, but, T. vulgaris extract was not as effective as the others. All three isolate were affected by these four plant extract with a minor differences.
The mean mutation frequency in the presence of extracts obtained from T. vulgaris and A. millefolium has been reduced to 21.7 × 10-9 and 9.8 × 10-9 respectively. In duplicated application of M. communis and T. polium extracts, we have not observed any mutant colony in agar media, though, bacterial cell suspension contained. 109 CFU.mL-1. Therefore, bacterial suspension containing 1011 CFU.mL-1 was used in these cases. After inoculation of this bacterial suspension on the agar media containing M. communis and T. polium extracts the mean mutation frequency values were 0.7 × 10-9 and 1.3 × 10-9 respectively.
5. Discussion
Successful eradication of H. pylori usually resulted in resolving the symptoms of its infection; moreover, it may have resulted in preventing the possible gastric cancer. Different eradication regimens have been developed in this respect. In the recent decades, the clarithromycin-based triple therapy was used as the recommended first-line empirical treatment of H. pylori infections (3, 5). Antibiotic resistance, especially clarithromycin resistance, is significantly increased all over the world. Therefore it is no longer recommended to follow empirical therapy of H. pylori infections in areas with high resistance rates (3, 15, 16). Point mutations in 23s rRNA gene was described as the main mechanism of clarithromycin resistance in H. pylori (17). The inhibition of these resistance-conferring mutations could serve as a novel strategy to overcome the antibiotic resistance issue. In recent years, a large number of literature reported the suggested anti-mutagenic agents in the medicinal plants (18-20).
Mutation is a complex process changing a genotype but not necessarily the phenotype. Therefore, the number of mutant colonies are significantly lower than the genotypically changed bacteria (11, 21, 22). We tracked the changes in the occurrence of mutation events which took place upon application of medicinal plants by means of “Mutation Frequency”. Mutation frequency shows the actual consequence of this intervention, because only the favorable mutation resulted in the appearance of an antibiotic resistant phenotype are detected.
The results obtained by comparing the mean mutation frequency values in the presence and absence of plant extracts. It is shown that there are reductions in the mean mutation frequency values after application of M. communis, T. polium, A. millefolium and T. vulgaris this amounts were 97.4%, 95.2%, 63.7% and 19.6%, respectively. This was an interesting outcome which could explain the existence of certain mutation preventative potentials in these plant extracts. Our results reveal that the ethanolic extract from T. vulgaris was not so effective in reducing the mutation frequency of clarithromycin resistance. This is in contrast with the results of Wei and Shibamoto (23). Such differences may be attributed to essential oil effects in their experiments rather than the ethanolic extract which was used in our experiments. Aida Wannes et al. reported weak anti-oxidant and probably anti-mutational activities of essential oils extracted from M. communis (24). They used essential oils obtained from leaf; stem and flower of this herb but ethanolic extract of the seeds was used in our experiment which showed its potent anti-mutational activity. These results demonstrated that the efficacy of ethanolic extracts is higher than that of essential oils. This finding was also supported by Aida Wannes et al. (24). The results from previous studies reporting the anti-mutagenic activity and free radical-scavenging properties of these herbs supported our findings (18, 19, 25-29). The reduction effect of plant extracts on antibiotic resistance may justifies the usage of these medicinal plant extracts.
Some appropriate experiments under in vivo condition on animal models or human volunteers are needed to confirm these findings. In this respect, the medicinal plant derived substances in comparison to the chemical agents could obtain more patients compliance and ease in designing of clinical trials in human volunteers. The extracts obtained from the tested herbs, beside anti-mutational effects had reported anti-inflammatory and protective effect on gastric mucosal layer (30). Such valuable findings confirmed the beneficial use of those in traditional herbal medicine for treatment of gastrointestinal discomforts especially those caused by H. pylori. The accumulative results obtained from such investigations could lead to the development of more effective eradication regimens containing medicinal plants in combination with antibiotics. This may even be more beneficial if the actual effective ingredients from such plant extracts are recognized and the most suitable is to be added to the present antibiotic regimens.
