Acidophilic and Acid Tolerant Actinobacteria as New Sources of Antimicrobial Agents against Helicobacter Pylori

1. Introduction

Helicobacter pylori is a gram-negative, microaerophilic bacterium that persistently colonizes the epithelium of human stomach cells. According to a comprehensive global survey, approximately 4.4 billion H. pylori infections were found around the world, with the distribution of 24.4% in Oceania to 70.1% in Africa (Hooi et al., 2017).

The number of diseases potentially contributed by H. pylori is increasing. They include gastric and extragastric diseases, such as dyspepsia, gastritis, gastric and colorectal cancers, laryngeal/pharyngeal cancers, lymphomas, iron-deficiency anemia, idiopathic thrombocytopenic purpura; some diseases in the skin, eyes, ears, nose, throat, liver, gallbladder, pulmonary, cardiovascular; diabetes, neuromyelitis optica, multiple sclerosis; and autoimmune, neurodegenerative, and pregnancy diseases (Testerman and Morris, 2014).

It should be mentioned that H. pylori can be treated by multiple antibiotics therapy; however, its resistance to commonly used antibiotics is increasing globally (Testerman and Morris, 2014). Therefore, it is needed to find new antimicrobial sources for its treatment.

Actinobacteria are among the most microbial sources of bioactive metabolites and have been used as a very useful source of antibiotics. Actinobacteria produce 130 out of ~190 clinical antibiotics (Hamedi et al., 2017), including broad-range antibiotics (e.g. tetracycline by Kitasatospora auerofaciens) and narrow-range antibiotics (e.g. erythromycin by Saccharopolyspora erythraea). Given the decreasing rate of the discovery of novel antibiotics, extremophilic microorganisms, like thermophiles, which have been poorly investigated, seem to be good sources for novel and effective antibiotics (Giddings and Newman, 2015).

Acidophilic actinobacteria are among common residents in acidic habitats, such as acidic soils, and grow at pH values of 3.5-6.5, with an optimal pH of ~4.5. Acid-tolerant actinobacteria grow at pH values of 4.5-7.5 with optimal growth at 5.0-5.5 pH range. Acidophilic and acid-tolerant actinobacteria have been studied as promising strains in biotechnology. For example, bactericidal (butalactin), fungicidal-insecticidal (nikkomycin), and antiprotozoal (granaticin A) activities were reported in Streptomyces corchorusii (Ueki and Kinoshita, 2004), Streptomyces tendae (Ginj et al., 2005), and Streptomyces lateritius, respectively. However, there is no report on the activity of acidophilic and acid-tolerant actinobacterial metabolites against H. pylori. In this regard, this study aimed to isolate these actinobacteria from acidic soils and investigate their activities against H. pylori.

2. Material and Methods

2.1. Microbial Test Strains

The H. pylori strain TUMS 10 was obtained from Medical Bacteriology Lab (Dept. of Pathobiology, School of Public Health, Tehran University of Medical Sciences , Tehran, Iran). Other bacteria and fungi that were used as test strains in antimicrobial assays were obtained from the University of Tehran Microorganisms Collection (UTMC) and included Escherichia coli UTMC 1465, E. coli TolC UTMC 1462, Pseudomonas aeruginosa UTMC 1463, Micrococcus luteus UTMC 1461, Mucor hiemalis UTMC 5057, Chromobacterium violaceum UTMC 1466, Candida albicans UTMC 5055, Staphylococcus aureus UTMC 1467, Pichia anomala UTMC 5056, and Bacillus subtilis UTMC 1464.

2.2. Microbial Culture Media

2.2.1. Helicobacter pylori Medium

Brucella agar (45 g/l) was enriched by defibrinated sheep blood (10% v/v) and fetal bovine serum (20% v/v). To reduce contamination, vancomycin (10 µg/ml), trimethoprim (5µg/ml), and amphotericin (5 µg/ml) were added to the medium.

2.2.2. Media for Isolation of Actinobacteria

It consisted of starch casein agar, compost agar, and rice bran agar (Hamedi et al., 2019). All media contained 15 g/l agar with pH 5.0±0.1 and were supplemented by amphotericin B (25 µg/ml) to inhibit the growth of fungi.

2.2.3. Medium for Purification and Short-time Maintenance of Actinobacteria

The ISP2 medium included malt (5 g/l), glucose (2 g/l), yeast extract (2 g/l), CaCO3 (1 g/l), and agar (15 g/l).

2.3. Antibiotics

Amphotericin B (Cipla Ltd, India), vancomycin (Sigma Co., USA), trimethoprim (Sigma Co., USA), and amphotericin (Sigma Co., USA) were used for the prevention of contamination or as control in the antibacterial assay.

2.4. Soil Sampling and Pretreatment

The samples were collected from acidic soils obtained from tea and rice farms located in the semitropical strip in the north of Iran. The soil samples were collected at the depth of 10-25 cm and transported to the laboratory in less than 24 h. After air-drying, crushing, and sieving the soil samples, three pretreatments were performed on them, including heat treatment at 120 ˚C for 10 min in an oven, exposure to electromagnetic waves (800 Hz, three min), and centrifuging (2950 g for 15 min, and the supernatant was used for actinobacterial isolation) (Hamedi et al., 2019).

2.5. Isolation and Identification of the Acidophilic Actinobacteria

The treated samples were serially diluted and appropriate dilutions were cultured on the media for isolation of acidophilic actinobacteria. The plates were incubated at 28 °C for 14 days. Putative actinobacterial colonies were sub-cultured on ISP 2 medium. The isolates were deposited in UTMC.

2.6. Determination of pH Tolerance of the Actinobacteria

To study the pH tolerance of the isolates, they were cultured in ISP-2 broth in various pH levels (1-14) and incubated at 28 oC for 14 days. Afterward, the turbidity of the broth was compared with that of the uninoculated ISP-2 broth and its increase was considered as growth and tolerance of the strain towards the related pH.

2.7. Preparation of Actinobacterial Metabolites

The isolates were cultured on the 100 ml screw-capped bottles containing ISP 2 agar slant (pH 5.5) as a sporulation medium. Appropriate concentrations of spores (~107-108/ml) were added to the 500 ml Erlenmeyer flask containing 50 ml seeding medium and incubated at 28 oC and 180 rpm for 48 h.

The seeding material (10 v/v) was added to the 1000 ml Erlenmeyer flask containing 150 ml fermentation medium and incubated at 28 oC and 180 rpm for 196 h. The fermentation material was centrifuged at 4000 rpm for 20 min to remove the biomass, and the supernatant was extracted by an equal volume of ethyl acetate. The organic phase (containing actinobacterial metabolites) was evaporated by a rotary evaporator at 38 oC at reduced pressure.

The obtained metabolite was weighed and divided into two screw-capped vials that were preserved at -20 oC. One vial was used for well agar diffusion assay as described below, and the other vial was mixed in methanol and carefully dissolved by using an ultrasonic bath for 2-3 min. Appropriate concentrations of the methanol-dissolved metabolites were poured on the blank paper disks that were put in the bottom of porcelain immunological plates. After evaporation of the solvent, the dried disk papers containing the metabolites were preserved at -20 oC in screw-capped vials.

2.8. Culture of Helicobacter pylori as an Inoculation Material

The H. pylori TUMS 10 was inoculated in a 50 ml Falcon tube containing 40 ml H. pylori medium (HP medium) and incubated at 37 oC in a CO₂ incubator (10 % CO₂) for 72 h. The tube was centrifuged at 3000 rpm for 5 min. An appropriate volume of HP broth was added to the bacterial sediment to achieve an appropriate concentration of H. pylori (~6×10⁸ colony forming unit [CFU]/ml). Finally, the ~1×10⁵ CFU/ml concentration of H. pylori was used in agar well diffusion or broth dilution methods.

2.9. Primary Screening of the Isolates for Antimicrobial Activity against H. pylori

Antimicrobial activity of the isolates against H. pylori was evaluated by agar diffusion methods using soluble extracts. Briefly, H. pylori inoculation material was spread onto the bacteriological plates containing HP agar. The metabolite disks were put on the surface of the plate or various concentrations of the metabolites were applied into the agar well. The plates were incubated in a humid and microaerophilic atmosphere at 37 oC for 3-4 days. Clarithromycin, amoxicillin, and metronidazole were used as controls.

Furthermore, the activity of the actinobacterial metabolites was studied against E. coli UTMC 1465, E. coli TolC UTMC 1462, P. aeruginosa UTMC 1463, M. luteus UTMC 1461, M. hiemalis UTMC 5057, C. violaceum UTMC 1466, C. albicans UTMC 5055, S. aureus UTMC 1467, P. anomala UTMC 5056, and B. subtilis UTMC 1464 by agar dilution method (Hamedi et al., 2019).

2.10. Determination of Minimum Inhibitory Concentrations of the Metabolites

The minimum inhibitory concentrations (MIC) of the selected metabolites (0-1000 µg/ml) against H. pylori were determined by the broth microdilution method (Meletiadis et al., 2010).

2.11. Combinatorial Effects of Actinobacterial Metabolite-antibiotics

The synergism or antagonism of the combination of actinobacterial metabolites and the antibiotics was performed by the checkerboard technique (Meletiadis et al., 2010). For this purpose, stock solutions and serial twofold dilutions of the antibiotics, including clarithromycin, amoxicillin, and metronidazole were prepared according to the protocols of the National Committee for Clinical Laboratory Standards.

Moreover, serial dilutions of the antibacterial metabolites were prepared as described above. Each one of the 24 wells on the microplate was filled by the molten HP medium. When the medium was solid, each well was inoculated with the inoculation material (0.1 mL) and then incubated for 72 h at 37 °C with 10% CO₂. The interaction was assessed by determining the fractional inhibitory concentration (FIC), which is the MIC of each antibacterial in combination divided by the MIC of each antibacterial when used alone. An FIC index lower and higher than one was considered as synergy and antagonism, respectively (Meletiadis et al., 2010).

2.12. Molecular Identification of Acidophilic Actinobacteria

Molecular identification of the actinobacterial isolates was performed by PCR amplification of 16S rRNA gene using universal primers 9F (5AAGAGTTTGATCATGGCTCAG-3) and 1541R (5-AGGAGGTGATCCAACCGCA-3) and their subsequent sequencing (Hamedi et al., 2019). The actinobacteria were cultured in BHI broth medium and incubated at 28 ˚C for 48 h. The biomass was obtained by centrifugation at 4000 rpm for 10 min, and the genomic DNA was extracted using a DNA extraction kit (Pooya Gene Azma Co., Tehran, Iran) and amplified. It should be noted that the PCR products were sequenced by Macrogen Inc. (South Korea). The sequences were blasted in National Center for Biotechnology Information (NCBI) and EzTaxon data bank, and finally, the sequences were submitted to GenBank (NCBI).

2.13. Brine Shrimp Lethality Bioassay

To find the toxicity of the metabolites, brine shrimp lethality bioassay was used by applying various concentrations of bioactive metabolites (200, 100, 50, and 25 µg/ml) to Artemia salina (Salimi et al., 2018).

2.14. High-performance Liquid Chromatography of Bioactive Metabolites

The metabolites obtained after extraction of the fermentation broth of the bioactive strains against H. pylori were analyzed by high-performance liquid chromatography (HPLC)-UV. Reversed-phase HPLC experiments were performed using XBridge C18 column 100×2.1 mm (Waters), 3.5 μm, solvent A (H2O−acetonitrile [95/5], 5 mmol NH4 acetate, 0.04 mL/L CH3COOH), solvent B (H2O−acetonitrile [5/95], 5 mmol NH4 acetate, 0.04 mL/L CH3COOH), and gradient system with 10% B increasing to 100% B in 30 min with a flow rate of 0.3 mL/min at 40 °C.

2.15. Chemical Constituents of the Bioactive Extract Using Gas Chromatography-mass Spectrometry Analysis

The chemical ingredients of the metabolites of selected strains were analyzed using GC-MS (Almasi et al., 2018).

3. Results

In total, 33 isolates were obtained from 83 acidic soil samples (pH 3.9-6.8). It should be noted that 20 and 13 of the isolates were obtained from the samples with pH levels of 3.9-5.0 and 5.4-6.8, respectively. Moreover, 25 and 8 isolates were collected from tea and rice farms, respectively. Table 1 summarizes the results of the molecular identification, sources of the isolates, and their related accession No. in Genbank. The majority of the isolates (91%) belonged to the Streptomyces genus while three of them (7%) belonged to the Kitasatospora genus. Some of the isolates were non-actinobacteria, including Methylobacterium and Varivorax; they were removed from the research. It is noteworthy that both of these isolates belonged to the Proteobacteria phylum.

3.1. pH Profile of the Isolates

Table 2 tabulates the results of the growth of the isolates in ISP-2 broth. It must be noted that 12% of the isolates, including Streptomyces sp. UTMC 3061, Streptomyces sp. UTMC 3218, Kitasatospora sp. UTMC 3221, and Streptomyces cirratus UTMC 3318, showed maximum growth at a pH level of less than 7 and were considered acidophiles.

Moreover, four strains (12%), including Streptomyces sp. UTMC 3019, Streptomyces sp. UTMC 3028, Streptomyces sp. UTMC 3055, and Streptomyces pratensis UTMC 3217 could grow at a higher pH than eight and were considered as alkaliphiles. The minimum pH for the growth of the strains was related to the Streptomyces sp. UTMC 3320.

3.2. Antimicrobial Susceptibility Profile of Helicobacter pylori

The H. pylori TUMS 10 used in the research was susceptible to clarithromycin, amoxicillin, and metronidazole in broth microdilution assay. Its minimum inhibitory concentrations among the aforementioned antibiotics were 0.5, 1, and 8 μg/ml, respectively.

3.3. Anti-Helicobacter pylori Activity of Actinobacterial Isolates

When the agar dilution method was used, no activity against H. pylori was observed in metabolite disks of actinobacterial isolates. However, in the broth microdilution method, 10 out of 33 isolated strains showed anti-H. pylori activity, including, Streptomyces sp. UTMC 3019, Streptomyces sp. UTMC 3026, Streptomyces coelicoflavus UTMC 3052, Streptomyces sp. UTMC 3061, Streptomyces sp. UTMC 3066, Streptomyces sp. UTMC 3211, Streptomyces sp. UTMC 3312, Streptomyces mirabilis UTMC 3317, S. cirratus UTMC 3318, and Streptomyces sp. UTMC 3320. Among them, two strains, namely Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318, were selected for secondary screening due to higher antibacterial activity and the results of their MICs against H. pylori were 125 and 62.5 µg /ml, respectively.

3.4. Checkerboard Analysis

Table 3 summarizes the results of the combinatorial effects of the selected actinobacterial metabolites and clarithromycin, amoxicillin, and metronidazole. As can be seen, the metabolites of Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318 at concentrations of 32 and 62.5 µg/ml, respectively, reduced the MIC of H. pylori to clarithromycin from 1 ug/ml to 0.5 µg/ml. However, no synergism was observed between the metabolites of the studied actinobacterial strains and amoxicillin and metronidazole since the actinobacterial metabolites did not reduce the MIC of these antibiotics from 1 and 16 µg/ml, respectively.

3.5. Other Biological Activities of the Isolates

3.5.1. Toxicity of the Metabolites

Toxicity of Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318 metabolites against brine shrimp indicated that the metabolites of both strains had no toxicity at concentrations of less than 32 µg/ml. Metabolites of Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318 at 125 and 62.5 µg/ml had 30% and 15% toxicity against A. salina, respectively.

3.5.2. Other Antimicrobial Activities of the Isolates

Among 33 actinobacterial isolates, 9 strains (27.3%) had other antimicrobial activities. More specifically, 6, 1, 1 and 3 isolates had antimicrobial activities against M. luteus UTMC 1461, B. subtilis UTMC 1464, P. anomola, and C. albicans, respectively (Table 4). No antimicrobial activity was observed against E. coli UTMC 1465, E. coli TolC UTMC 1462, P. aeruginosa UTMC 1463, M. hiemalis UTMC 5057, C. violaceum UTMC 1466, and S. aureus UTMC 1467 by agar dilution method.

3.5.3. Thin-Layer Chromatography–bioautography of Selected Metabolites against H. pylori

No inhibition zone was observed around the thin-layer chromatography plates of the metabolites obtained from the strains, Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318.

3.5.4. Chemical Analysis of the Metabolites

According to the results of HPLC analysis, there were four and six UV-active compounds in the fermentation broth extracts of the strains Streptomyces sp. 3061 UTMC (Figure 1A) and S. cirratus UTMC 3318 (Figure 1B) at the wavelengths of 254 and 366 nm, respectively. Figure 2 shows the results of the GC-MS analysis of the metabolites of the selected strains that were obtained after ethylacetate extraction.

Figure 1. Fractionation of the metabolites of Streptomyces sp. UTMC 3061 (A) and Streptomyces cirratus UTMC 3318 (B) by HPLC at wavelengths 254 and 366 nm

Figure 2. The chemical constituents of the metabolites of Streptomyces sp. UTMC 3061 (A) and Streptomyces cirratus UTMC 3318 (B) found by gas chromatography and mass spectrometry analysis

Analysis of the results of the metabolites of Streptomyces sp. 3061 UTMC and S. cirratus UTMC 3318 led to five and four compounds, respectively. Metabolites of Streptomyces sp. 3061 UTMC consisted of carbamic acid (retention time : 5.788 min), maltol (RT: 8.182 min), 2.4-di-tert-butylphenol (RT: 13.549 min), methyl dimendone (RT: 18.094 min), and oleamide (RT: 22.233 min). Moreover, the metabolites of S. cirratus UTMC 3318 contained maltol (RT: 8.191 min), 2.4-di-tert-butylphenol (RT: 13.548 min), prolylleucyl (RT: 18.268 min), and oleamide (RT: 22.171 min).

4. Discussion

Previous studies have noted the importance of acidophilic microorganisms in biotechnology. This study aimed to assess the importance of acidophilic actinobacteria in antimicrobial production against H. pylori. All actinobacteria isolated in this study were obtained from tea and rice fields with pH levels of 3.9-6.8 and should be considered acid tolerant. Moreover, 12% of the strains had maximum growth at acidic conditions and were acidophiles.

Another important finding was that 76% of the strains were isolated from the tea field, and the average pH of the tea fields was 0.5 units less than that of the rice fields. All three Kitasatopsora isolates were obtained from the tea field with an average pH of 4.36. It can be concluded that tea-field soils are better sources for isolation of acidophilic actinobacteria than rice-field soils. However, the actinobacteria isolated from tea fields did not show any antibacterial activity against H. pylori. All of the 10 active actinobacterial isolates belonged to the Streptomyces genus, including Streptomyces sp. 3061 UTMC, and S. cirratus UTMC 3318.

Streptomyces sp. 3061 had 99.41% similarity to Streptomyces spinoverrucosus obtained from National Brain Research Centre with the code NBRC 14228 (T). Until now, two strains of S. spinoverrucosus have been reported, namely S. spinoverrucosus Diab 163MAT (NBRC 14228=NCIB 11666) (Diab and Al-Gounaim, 1982) and S. spinoverrucosus SNB-032.

The first strain was the type strain isolated from air in Kuwait, while the second strain was isolated from a marine sediment sample collected from Trinity Bay, Texas, USA and had 99% similarity in 16S rRNA gene sequence to type strain (Hu et al., 2012). From the second strain, three anthraquinones were discovered, including galvaquinones A-C (Hu et al., 2012); however, no antibacterial activity was reported in galvaquinones.

The S. cirratus UTMC 3318 had a 100% similarity to S. cirratus obtained from Northern Regional Research Lab with the code B-3250T. S. cirratus 248-Sq2 that was isolated from the soil in Norikura highland, Japan, and produced cirratiomycin A, cirramycin A, and cirramycin B that were active against Lactobacillus casei and some strains of Streptococcus and Mycobacterium (Shiroza et al., 1982). The S. cirratus F2-2 that was isolated from a banana plantation in Zhangzhou, China had good antibacterial activity against some gram-negative bacteria, including Pseudomonas putida, P. fluorescens, Burkholderia sepacia, and Escherichia coli (Shirokikh et al., 2018).

The most obvious findings that emerged from the chemical analysis of the metabolites of Streptomyces sp. UTMC 3061 and S. cirratus UTMC 3318 was that both of them produced maltol, 2.4-di-tert-butylphenol, and oleamide.

Maltol is a naturally occurring organic compound in plants and is used primarily as a flavor enhancer in bread and cake (Han et al., 2015). This is in line with the results of previous studies which have indicated that maltol is also produced by various members of actinobacteria (Kornsakulkarn et al., 2014).

Some antibiotic activities were reported from maltol derivatives (Salsbury et al., 2015). It should be noted that 2,4-di-tert-butylphenol is an alkylbenzene and a member of phenols that are produced by actinobacteria with antibacterial and antifungal activities (Belghit et al., 2016). Oleamide is an amide of oleic acid and occurs naturally in plants and animals and has antimicrobial activity (Shao et al., 2016).

Streptomyces sp. 3061 UTMC also produced carbamic acid and methyl dimendone. Carbamic acid had various biological activities, including antibacterial activity against Mycobacterium tubercolosis (Zanatta et al., 2006) and methicillin-resistant S. aureus (Han et al., 2004) and inhibition of histidine acetylase (Rayudu, 1990). H. pylori produced carbamic acid by affecting its urease on urea. It should be mentioned that urease can speed up carbamic acid production (by hydrolysis of urea) or consumption (by the synthesis of urea) (Zimmerli and Schlatter, 1991).

Given the vital importance of urease as the main shield of H. pylori from gastric, an increase in the carbamic acid may decrease the activity of urease and help the removal of H. pylori from the stomach. It should also be noted that 2-Methyldimedone or 1,3 cyclohexanedione was found in essential oils of some leptospermone plants and has weak antibacterial activity against Clostridium difficile and Clostridium perfringens (Jeong et al., 2009).

Prolylleucyl is another metabolite that was detected in the extract of S. cirratus UTMC 3318. This is also consistent with our earlier observations which indicated the antimicrobial activity of this dipeptide that had been isolated as a metabolite of actinobacteria (El Euch et al., 2018).

Based on the findings, it can be said that acidophilic actinobacteria can be promising sources of active metabolites against H. pylori. The metabolites of S. cirratus UTMC 3318 and Streptomyces sp. UTMC 3061 showed limited toxicity against Eukaryotes. They also showed good synergism with clarithromycin, a current medication used for H. pylori treatment. This finding suggests that these acidophilic actinobacteria can be good sources of active metabolites. Therefore, future studies on the current topic are recommended to define the details of the chemical composition of the metabolites and compare their effects with those of pure compounds obtained from plant or animal sources or chemical synthesis.

Authors' Contribution

Study concept and design: J. H.

Acquisition of data: L. E.

Analysis and interpretation of data: J. H. and R. B.

Drafting of the manuscript: J. H.

Critical revision of the manuscript for important intellectual content: J. H.

Statistical analysis: L. E.

Administrative, technical, and material support: J. H., Gh. Z. and R. B.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Conflict of Interest

The authors declare that they have no conflict of interest.

Grant Support

This study was financially supported in part by University of Tehran, Grant No. 66104001/D6/046.

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