1. Background
Resistance to antibiotics is a ubiquitous relentless clinical problem. New antibiotic development by pharmaceutical sector has aggravated the challenge of widespread resistance and signals a critical need for innovation (1). Cyanobacteria have a wide range of secondary metabolites including antibiotics (2). Researches to identify antimicrobial compounds produced by cyanobacteria, have recently received attention as a new source of novel antimicrobial substances. Ethyl acetate extract of Spirulenaplatensis can inhibit some Gram positive and Gram negative bacteria and Candida albicans (3). Oscillatoria spp. can produce derivatives with antimicrobial activity (4). Nowadays, combinations of antimicrobials are usually used in medicine to broaden antimicrobial spectrum and generate synergistic effects. Drug combinations have potentials for counteracting drug resistance (1). The economical and oligotrophic growth (5-7) of Leptolyngbya sp., form the aim of this research, whereas arriving at new antimicrobial compounds is of prime importance.
The current research studies the antimicrobial effects of oligotrophic Cyanobacteriumleptolyngbya sp. independently and in synergy with antibiotics. This taxonomic cluster was originally named as “LPP−group B” by bacteriologists (8), and has been recently supported and accepted by molecular analysts (9). This cyanobacteria is moderately a new thermophilic halophile or halotolerant genus (10, 11). The basic and fundamental requirements for cyanobacterial biotechnology are the availability of natural cyanobacterial sources and understanding their ecobiological properties. Therefore, a detailed study of its habitats was made to identify the available cyanobacterial species and subsequently to isolate, purify and establish a culture collection which could be used for further studies (12).
The successful cultivation and growth of cyanobacteria require modification in media composition (9). Several liquid media have been modified for the culture of marine and freshwater species e.g., artificial seawater nutrients III (ASN-III), Marine Medium (MN), and Blue-green 11 (BG-11) (8). The modification to find a desirable culture related to requirements of cyanobacteria may differ from species to species (13). In the current research, the optimum growth conditions of Leptolyngbya sp. were studied. BG-11 medium is a general culture medium for cyanobacteria. BG-11medium was subject to some modifications in Nitrogen, phosphorus, magnesium, iron and copper contents. The results are discussed.
2. Objectives
The current study aimed to access new natural antimicrobial compounds; therefore, the current research studied antimicrobial properties of Leptolyngbya sp..
3. Materials and Methods
3.1. Sampling and Culturing
The unexplored fresh or hot benthic waters in East Azerbaijan province of Iran were the sampling regions. Samples were taken directly by immersion in water of sampling bottles by "grab" or "snap" sampling methods (14). The samples were stored at a temperate range of 10-17°C under dark conditions in a storage box in order to decrease metabolism and to avoid the growth interfering factors during sampling and transportation.
3.2. Growth Conditions
The samples were cultured batch wise in BG-11 agar and broth media (15) and illuminated at a light intensity of 3500 lux (16 hours light: 8 hours dark) at 22°C and shaking at 100 rpm. The samples were screened for Leptolyngbya sp. by different purification methods according to the degree of contamination of samples.
3.3. Molecular Analysis
DNA extraction with boiling method For PCR test was performed and then was amplified and electrophoresis (16). PCR products were sequenced and aligned with the cited sequences in NCBI.
3.4. Oligotrophic and Halophilic or Halotolerant Aspects of the Leptolyngbya sp.
Growth of Leptolyngbya sp. in oligotrophic state was studied in the CHU NO.10 modified medium. The medium contains very low amounts of nutrients and salts and is therefore only suitable for oligotrophic strains. Halophilic or halotolerant properties of Leptolyngbya sp. were tested with van Rijn and Cohen (RC) Medium (17). RC modified media were prepared at 1/3 and 2/3 reduced salt concentrations compared to the original.
3.5. Antibacterial and Antifungal Bioassay
3.5.1. Extract Preparation
Leptolyngbya sp. strains grown in the liquid medium were harvested at late exponential phase. The biomass was extracted with hexane, methanol, diethyl ether, ethyl acetate, chloroform organic solvents. Aqueous crude extracts were also used.
3.5.2. Antimicrobial Activity on Test Microorganisms
The following bacteria and fungi were used as test organisms: Staphylococcus aureus (PTCC 1112), Micrococcus luteus (PTCC 1625), Escherichia coli (PTCC 1329), Erwiniacarotovora (PTCC 1675), Salmonella typhi (PTCC 1609) and Candida kefyr (ATCC 38296).
Minimum inhibitory concentrations (MIC), Minimum bactericidal inhibition (MBC) and minimum fungicidal inhibition (MFC) were assayed by broth dilution methods (18, 19). The antibacterial effects of a combination of the chloroform extract, which exhibited the highest antimicrobial activity with antibiotics, were assessed by the checkerboard test and fractional inhibitory concentration (FIC) (20). Where more than one combination resulted in a change in the MICs value of the extract or antibiotic, the FICI was described as the total of the individual FIC values (20).
3.6. Evaluation of the Effects of BG-11 Medium Components on the Optimum Growth and Antimicrobial Activity of C. leptolyngbya BG-11 medium components
(0.75 mg) MgSO4.7H2O, (36 mg) CaCl2.2H2O, (1500 mg) NaNO3, (30 mg) K2HPO4, (20 mg) Na2CO3, (1 mg) Na2EDTA.2H2O, (6 mg) Ferric ammonium citrate, (1 ml) Micronutrient solution (A5+CO) (8), final volume 1000 ml with deionized water and pH after filtrate ~7.1.
(A5+CO) components: (286 mg) H3BO3, (181 mg) MnCl2.4H2O, (22 mg) ZnSO4.7H2O, (39 mg) NaMoO4.2H2O, (79 mg) CuSO4.5H2O in the final volume 100 ml with deionized water. Culture media was prepared in three forms: 0, 1.5 and 2 fold of BG-11 salts. Incubation conditions were as described earlier. After 13 days, the culture media were taken for growth measurement, determination of dry weight and chloroform extracts antibacterial assay.
3.7. Growth Measurements
Chlorophyll-a, from each sample was extracted in 80% acetone and the content of the pigments was determined using the procedure of Myers and Kratz at an absorbance of 663 nm. Specific growth rate was calculated using the formula: µ= [ln (Bf/Bi)]/ti. Where Bi is the initial chlorophyll-a, and Bf is the chlorophyll-a, at the end of incubation. Productivity was calculated from the formula: P = (Xi-X0)/ti, where P = productivity (mg L-1day-1), X0 = initial biomass density (mg L-1), Xi = biomass density at time i (mg L-1) and ti =time interval (h) between X0 and Xi (21).
3.8. Determination of dry Weight
10 ml from different cultures were washed several times with distilled water to remove soluble salts. The cyanobacteria cells were then dried at 80ºC for 30 min and weighed (22).
4. Results
4.1. Isolation
Overall, 35 samples were isolated out of which only 2 samples were Leptolyngbya spp.
4.2. Identification
The 16S rRNA sequences of two isolates from Heravi location of Mehranrud and Bostan Abad underground hot spring were subject to NCBI blast. The former corresponded to Leptolyngbyanodulosa UTEX 2910 with accession number (gi|119367623|EF122600.1), 99% identity and 0% gap. The latter corresponded to Leptolyngbya spp. 0418 with accession number (gi|15842033|EU068735.1), 99% identity and 0% gap.
4.3. Growth Ability
B-110 culture medium is nitrogen limited medium. The isolated strains of C. leptolyngbya were grown in the culture medium and therefore may be considered as putative nitrogen fixing species for C. leptolyngbya isolates. These strains relieve suitable growth in the CHU-10 modified medium. Bottle-green and glucose pigments in nitrogen limited media, namely BG-110 and CHU-10 modified culture did not occur. Among the different dilutions of saline RC culture medium, the best growth was observed at 1:3 dilution. Available strains have growth ability in the RC 1/3 modified.
4.4. Antimicrobial Results
Chloroform extracts compared to other solvents, showed better results in antimicrobial testing. Therefore, further investigation of the antimicrobial activity with chloroform extract was performed. The Screening tests showed that Gram positive bacteria were more susceptible than Gram negative bacteria. The antimicrobial activities are given in Table 1.
Table 1.Antimicrobial Activity of the Chloroform Extracts of Leptolyngbya sp. 1 For Bacteria and Leptolyngbya sp. 2 for Fungi Alone and Different Antibiotics Combined.
| Strains | Agent | MIC, µg/mL | FIC | FICI | Outcome | |
|---|---|---|---|---|---|---|
| Alone | Combined | |||||
| Micrococcus luteus | Leptolyngbya sp. 1 Gentamicin | 80/>80 a 1.2 | 20 (0.3) | 0.25 (0.25) | 0.5 | Synergistic |
| Micrococcus luteus | Leptolyngbya sp. Erythromycin | 80/>80 (0.3) | 20 (0.15) | 0.25 (0.5) | 0.75 | Additive |
| Staphylococcus aureus | Leptolyngbya sp. 1 Gentamicin | 40/80 (0.6) | 20 (0.15) | 0.5 (0.25) | 0.75 | Additive |
| Staphylococcus aureus | Leptolyngbya sp. 1 Erythromycin | 40/80 (0.3) | 20 (0.15) | 0.5 (0.5) | 1 | Additive |
| Salmonella typhi | Leptolyngbya sp. 1 Gentamicin | Resistance (1.2) | 20 (0.6) | 0 (0.5) | 0.5 | Synergistic |
| Salmonella typhi | Leptolyngbya sp. 1 Ceftazidime | Resistance (12.5) | 20 (6.2) | 0 (0.5) | 0.5 | Synergistic |
| Candida kefyr | Leptolyngbya sp. 2 Amphotericin B | 40/60 (0.6) | 20 (0.3) | 0.5 (0.5) | 1 | Additive |
aThe fractions are related to the MBC data of C. leptolyngbya sp. 1 and MFC data of C. leptolyngbya sp. 2.
Table 2 shows the values of maximum specific growth rate (µmax) and productivity (P312) 13 days after culturing two strains of Leptolyngbya genera at different salt concentrations of BG-11 culture media. Their antibacterial effects were evident against M. luteus . Due to the importance of nitrogen, phosphorus, iron, magnesium and copper ions in the NaNO3, K2HPO4, Ferric ammonium citrate, MgSO4, CuSO4 salts, have been studied in the current research ( Table 2). With the exception of phosphorus and copper salts, high concentrations of other elements did not lead to increased antibacterial activity. None of the isolated C. leptolyngbya strains showed signs of growth at two-fold increased micronutrients solution (A5+CO) ( Table 2).
Table 2.Growth Ability With µ max , P 312 Tests and Antibacterial Activity Research With Kirby-Bauer Disk Diffusion Method
| Treatments | µmax, daya | P312, mgLadaya | Inhibition Zonesb |
|---|---|---|---|
| BG-11 plus 0.5 fold of K2HPO4 salt to Leptolyngbyasp. 1 | 0.178 ± 0.004 | 9.93 ± 0.16 | Resistance |
| BG-11plus 1 fold of K2HPO4 salt to Leptolyngbya sp.1 | 0.224 ± 0.002 | 10.57 ± 0.27 | 9 ± 0.63 |
| BG-110plus 1 fold of K2HPO salt to to Leptolyngbya sp.1 | 0.088 ± 0.003 | 4.8 ± 0.75 | 8 ± 1.09 |
| No K2HPO4salt BG-11 to Leptolyngbya sp.1 | No growth | No growth | 14 ± 0 |
| BG-11plus 0.5 fold of K2HPO4 salt to Leptolyngbya sp.2 | 0.162 ± 0.004 | 6.89 ± 0.33 | Resistance |
| BG-11 plus 1 fold of K2HPO4 salt to Leptolyngbya sp.2 | 0.193 ± 0.003 | 4.32 ± 0.22 | Resistance |
| BG-110plus 1 fold of K2HPO4 salt to Leptolyngbya sp.2 | 0.098 ± 0.004 | 6.25 ± 0.18 | 9 ± 1.09 |
| No K2HPO4 salt BG-11 to Leptolyngbya sp.2 | No growth | No growth | 15 ± 0.89 |
| BG-11plus 0.5 fold of NaNO3 salt to Leptolyngbya sp.1 | 0.169 ± 0.003 | 7.37 ± 0.9 | Resistance |
| BG-11 plus 1 fold of NaNO3 salt to Leptolyngbya sp.1 | 0.194 ± 0.008 | 8.01 ± 0.1 | Resistance |
| BG-11plus 0.5 fold of NaNO3 salt to Leptolyngbya sp.2 | 0.157 ± 0.005 | 4.8 ± 0.24 | Resistance |
| BG-11 plus 1 fold of NaNO3 salt to Leptolyngbya sp.2 | 0.168 ± 0.006 | 5.6 ± 0.17 | Resistance |
| BG-11plus 0.5 fold of Ferric ammonium citrate to Leptolyngbya sp.1 | 0.131 ± 0.001 | 6.08 ± 0.79 | Resistance |
| BG-11 plus 1 fold of Ferric ammonium citrate to Leptolyngbya sp.1 | 0.155 ± 0.003 | 5.44 ± 0.18 | Resistance |
| No Ferric ammonium citrate BG-11 to Leptolyngbya sp.1 | 0.148 ± 0.005 | 5.44 ± 0.14 | Resistance |
| BG-11plus 0.5 fold of Ferric ammonium citrate to Leptolyngbya sp.2 | 0.13 ± 0.001 | 3.04 ± 0.58 | Resistance |
| BG-11 plus 1 fold of Ferric ammonium citrateto Leptolyngbya sp.2 | 0.179 ± 0.006 | 3.04 ± 0.35 | Resistance |
| No Ferric ammonium citrate BG-11 to Leptolyngbya sp.2 | 0.145 ± 0.002 | 1.76 ± 0.9 | Resistance |
| BG-11plus 0.5 fold of CuSO4. 5H2O salt to Leptolyngbya sp.1 | No growth | No growth | 14 ± 0 |
| BG-11 plus 1 fold of CuSO4. 5H2O salt to Leptolyngbya sp.2 | No growth | No growth | 10 ± 1.26 |
| BG-11plus 0.5 fold of CuSO4.5H2O salt to Leptolyngbya sp.1 | No growth | No growth | 11 ± 0 |
| BG-11 plus 1 fold of CuSO4. 5H2O salt to Leptolyngbya sp.2 | No growth | No growth | 9.5 ± 0 |
| BG-11 plus 0.5 fold of MgSO4. 7 H2O salt to Leptolyngbya sp.1 | 0.144 ± 0.003 | 4.16 ± 0.33 | 9 ± 0.63 |
| BG-11 plus 1 fold of MgSO4. 7H2O salt to Leptolyngbya sp.1 | 0.184 ± 0.005 | 4.807 ± 0.1 | 7.5 ± 0.37 |
| BG-11 plus 0.5fold of MgSO4. 7H2O salt to Leptolyngbya sp.2 | 0.133±0.008 | 2.4 ± 0.11 | 8 ± 0 |
| BG-11 plus 1 fold of MgSO.4. 7H2O salt to Leptolyngbya sp.2 | 0.188 ± 0.004 | 3.044 ± 0.98 | 7.5 ± 0.31 |
| BG-11 plus 0.5 fold of micronutrients [A5+CO] to Leptolyngbya sp.1 | 0.118 ± 0.001 | 2.24 ± 0.8 | Resistance |
| BG-11 plus 1 fold of micronutrients [A5+ CO] to Leptolyngbya sp.1 | 0.13 ± 0.005 | 1.76 ± 0.19 | Resistance |
| BG-11 plus 0.5 fold of micronutrients [A5+CO] to Leptolyngbya sp.2 | Cell death | Cell death | - |
| BG-11 plus 1 fold of micronutrients [A5+CO] to Leptolyngbya sp.2 | Cell death | Cell death | - |
| Routine BG-11 (Control) to Leptolyngbya sp.1 | 0.135 ± 0.004 | 2.24 ± 0.28 | 8 ± 0.7 |
| Routine BG-11 (Control) to Leptolyngbya sp.2 | 0.13 ± 0.001 | 1.76 ± 0.37 | 7.5 ± 0.31 |
5. Discussion
Leptolyngbya strains grew well in the CHU-10 modified medium. The findings were in support of those of Douterelo et al. (5), Mateo et al. (6) and Perona et al. (7) in which Leptolyngbya sp. was oligotrophic. In the modified RC, saline concentration was approximately equivalent to 34 psu. This means that cyanobacteria have ability for growth in the average saline sea waters. These observations are in agreement with those of Abed et al. (10), and Chatchawan et al. (11). According to the antimicrobial test, about 42.85% of all the interactions were synergistic, while indifferent and antagonistic interactions were not observed. Additive activity was against the Salmonella typhi. Synergistic activity between leptolyngbya sp.2 and Amphotericin B against C. kefyr shows that this strain possesses antifungal activity. Over all, strain 2 has more antifungal activity than strain 1. Antibacterial activity of strain 1 is higher than that of strain 2. Gram positive bacteria were more susceptible than Gram negative bacteria confirming Dixon et al. report (23).
Table 2shows that combination of antibiotics and extracts had better results than their single use. According to the first row of this table, MIC of the extract alone is 80 µg/mL; while MIC in combination with antibiotic has decreased to 20 µg/mL. MIC for antibiotic alone in this case is 1.2 µg/mL, while in combination with the extract it decreased to 0.3 µg/ml. The results demonstrated that the extracts combined with antibiotics significantly increased antimicrobial inhibition effects. Biomass increased with increasing concentrations of NaNO3, K2HPO4, Ferric ammonium citrate in µmax and productivity tests were considerable ( 24 ). Table 2 shows the importance of salts, particularly phosphate salt in the BG-11 culture medium for biomass production and growth of Leptolyngbya sp.. This is probably applicable to all Cyanobacteria. K2HPO4 is so important that its absence is quite clear.
The importance of phosphorus and potassium content along with the buffering capacity of K2HPO4 indicate that Cyanobacteria will not be able to grow in the absence of K2HPO4. This condition is also true with the lower concentration of nitrogen in the culture medium. The nitrogen limited BG-110 culture media was used. Yellowish pigments produced in this medium show a major reduction in µmax with chlorophyll-a test calculation. Contrary to this phenomenon, higher values in µmax test were observed as a result of high MgSO4 concentration showing increased chlorophyll-a pigment. Absence of growth at two-fold increased micronutrients solution (A5+CO)indicates toxic effect of the higher concentrations of the trace elements. Increased antimicrobial activity at higher concentration of MgSO4, could be attributed to its sulfur content (25).
Importance of phosphorus, and nitrogen for cyanobacteria growth is in agreement with the reports by Downing et al. (26), Reynolds (27), Rolland et al. (28), and Xieet al. (29). Increased concentration of copper salt and micronutrients solution (A5+CO) resulted in no growth promotion confirming Ruiter et al. work (30). According to antimicrobial test results, Leptolyngbya species have more antimicrobial effects against the Gram positive bacteria and Candida genera than the Gram negative or fungi; this could be due to hydrophobic property of Gram-negative bacteria outer membrane lipopolysaccharides. The results indicate that the increased antimicrobial activity and biomass production are reversely related.
