Isolation, Optimization, and Structural Characterization of Glycolipid Biosurfactant Produced by Marine Isolate Shewanella algae B12 and Evaluation of Its Antimicrobial and Anti-biofilm Activity.

Biosurfactants are microbial-derived compounds with surface and emulsifying activities. Environmental and industrial applications make glycolipid biosurfactants particularly interesting among the several categories of biosurfactants. A potential glycolipid biosurfactant resource, Shewanella algae, was isolated from marine samples at the Persian Gulf. The glycolipid biosurfactant caused a reduction in water surface tension from 72 to 43 mN/m with a 0.25 mg/mL critical micelle concentration (CMC). Two-level factorial design was then applied for optimization of biosurfactant production, where a maximal reduction of culture broth surface tension (31 mN/m) acquired in the presence of crude oil (0.5%, v/v), NaNO3 (0.2 g/L), NH4Cl (0.7 g/L), and peptone (0.5 g/L). GC-MS analysis of the culture broth showed when crude oil was used as the sole carbon source, S. algae was able to degrade most of its alkane components. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy revealed the glycolipid structure of biosurfactant. The glycolipid biosurfactant exhibited considerable growth inhibition of clinical bacterial pathogens and disrupted the preformed biofilms of Bacillus cereus (83%), Streptococcus pneumoniae (53%), Pseudomonas aeruginosa (92%), Escherichia coli (64%), Klebsiella pneumoniae (87%), and Acinetobacter sp. (72%). In conclusion, the glycolipid biosurfactant secreted by S. algae exhibited a wide range of functional properties and was evidenced as a promising candidate for biomedical application.

Study the antibacterial and antibiofilm activity of Carum copticum against antibiotic-resistant bacteria in planktonic and biofilm forms.

OBJECTIVES: Microbial biofilms has attracted interest in the recent years because they has become the most important cause of nosocomial infections. This study was aimed to examine the antibacterial activities of Carum copticum extracts on the development of microbial biofilms and planktonic form of six pathogenic bacteria. METHODS: Antimicrobial activity of the crude extracts against the planktonic form of six pathogenic bacteria: Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Klebsiella pneumonia was evaluated by using the disc diffusion method. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values was determined by macro-broth dilution technique. Anti-biofilm effects were assessed by microtiter plate method. The chemical composition of the herbal extract was identified by GC-MS. RESULTS: According to disc diffusion test (MIC and MBC) the ability of C. copticum extracts for inhibition of bacteria in planktonic form was confirmed. The best inhibitory effect of this plant on S. aureus and low inhibitory effect on A. baumannii in planktonic forms were observed. These extracts were efficient to inhibit biofilm structures and concentration of each extract has direct relation with inhibitory effect. The maximum and minimum inhibitory effects of C. copticum methanolic extract on biofilm formation were observed on A. baumannii (98%) and K. pneumoniae (19%) respectively. CONCLUSION: The GC-MS analysis revealed that five active compounds were present in the extract of this plant. Data obtained, suggested that the C. copticum extracts applied as antimicrobial agents against these pathogens particularly in biofilm making.

Antibacterial Activity of Semenovia suffruticosa (Essential Oil) Against Pathogenic Bacteria and Determination of Chemical Composition of Essential Oils by Gas Chromatography-Mass Spectrometry Analysis in Four Regions of Kerman.

The use of medicinal plants has been considered due to increasing bacterial resistance to antibiotics. Semenovia suffruticosa is a plant with high potential in medicine. The aim of this study was to evaluate the antibacterial activities and gas chromatography-mass spectrometry (GC-MS) analyses of the essential oil of S. suffruticosa from four regions in Kerman: Hezar Mountain, Laleh Zar, Bidkhan, and Rabor. The essential oil of this plant was extracted by Clevenger. The antibacterial activities were evaluated against three Gram-negative bacteria (P. aeruginosa, K. pneumonia, Acinetobacter) and three Gram-positive bactria (B. subtilis, S. aureus, S. pneumoniae). By disk diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined. The composition of essential oil was identified by GC-MS analysis. In our study, most yield of essential oil of S. suffruticosa was 1.2% from the Laleh Zar region and the main components were Z-ß-ocimene (25.4%) in Laleh Zar, linalool (17.7%) in Hezar, Z-ß-ocimene (18.5%) in Bidkhan, and ß-pinene (10.5%) in the Rabor region. Maximum inhibition zone by the essential oil from region Bidkhan was observed against K. pneumoniae (30 mm). The MIC value for S. suffruticosa collected from Laleh Zar was 1.25 mg/ml against S. aureus and S. pneumonia. The results of this study confirm that the significant antibacterial effects of S. suffruticosa and make it a valuable compound in essential oils for pharmaceutical use and a good replacement for chemical antibiotics. Environmental conditions can result in a difference in yields and components; this can be considered significant potential for this plant.

Antibacterial Activity of Euphorbia hebecarpa Alcoholic Extracts Against Six Human Pathogenic Bacteria in Planktonic and Biofilm Forms.

BACKGROUND: Biofilm formation is a primary cause of considerable bacterial destruction. OBJECTIVES: In an effort to combat these industrial and medical bacterial biofilm problems, our study aims to determine the antimicrobial effect of Euphorbia hebecarpa. MATERIALS AND METHODS: The inhibition efficiency of alcoholic extracts on the planktonic form of six pathogenic bacteria was evaluated using a disk diffusion technique. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values were determined by means of a macrobroth dilution method. The effects of the extracts on biofilms were calculated using a microtiter plate method. RESULTS: The results of the disk diffusion assay (MBC and MIC) confirmed that E. hebecarpa ethanolic extracts were more efficient than methanolic extracts in the inhibition of planktonic forms of bacteria. Also, the inhibitory effect of the extracts in a broth medium was greater than in a solid medium. Extracts of E. hebecarpa were found to inhibit biofilm formation better than demolish of biofilm and preventing metabolic activity of bacteria in biofilm structures. The greatest inhibitory effects of E. hebecarpa extracts were observed for the biofilm formation of B. cereus (92.81%). In addition, the greatest demolition was observed for the S. aureus biofilm (74.49%), and the metabolic activity decrement of this bacteria was highest (78.21%) of all the tested bacteria. CONCLUSIONS: The results of this study suggest that E. hebecarpa extracts can be used to inhibit the planktonic and biofilm forms of these selected bacteria.

Study the symbiotic crude oil-degrading bacteria in the mussel Mactra stultorum collected from the Persian Gulf.

Symbiotic associations are complex partnerships that can lead to new metabolic capabilities and the establishment of novel organisms. The diversity of these associations is very broad and there are still many mysteries about the origin and the exact relationship between the organisms that are involved in a symbiosis. The aim of the present study is to find symbiotic crude-oil degrading bacteria in the mussels that collected from the Persian Gulf. Fifteen crude-oil degrading bacteria were isolated from Mactra stultorum mussel that collected from oil contaminated area at Persian Gulf. According to high growth rate on crude oil five strains were selected from 15 isolated strains for more study. Determination of the nucleotide sequence of the gene encoding 16S rRNA show that these isolated strains belong to: Alcanivorax dieselolei strain BHA25, Idiomarina baltica strain BHA28, A. dieselolei strain BHA30, Alcanivorax sp. strain BHA32 and Vibrio azureus strain BHA36. Analysis of remaining of crude oil by Gas Chromatography (GC) confirmed that these strains can degrade: 64%, 63%, 71%, 58% and 75% of crude oil respectively.

Enrichment and isolation of crude oil degrading bacteria from some mussels collected from the Persian Gulf.

To date, little is known about existing relationships between mussels and bacteria in hydrocarbon-contaminated marine environments. The aim of this study is to find crude oil degrading bacteria in some mussels at the Persian Gulf. Twenty eight crude oil degrading bacteria were isolated from three mussels species collected from oil contaminated area at Persian Gulf. According to high growth and degradation of crude oil four strains were selected between 28 isolated strains for more study. Determination the nucleotide sequence of the gene encoding for 16S rRNA show that these isolated strains belong to: Shewanella algae isolate BHA1, Micrococcus luteus isolate BHA7, Pseudoalteromonas sp. isolate BHA8 and Shewanella haliotis isolate BHA35. The residual crude oil in culture medium was analysis by Gas Chromatography (GC). The results confirmed that these strains can degrade: 47.24%, 66.08%, 27.13% and 69.17% of crude oil respectively. These strains had high emulsification activity and biosurfactant production. Also, the effects of some factors on crude oil degradation by isolated strains were studied. The results show that the optimum concentration of crude oil was 2.5% and the best degradation take place at 12% of salinity. This research is the first reports on characterization of crude oil degrading bacteria from mussels at Persian Gulf and by using of these bacteria in the field the effect of oil pollution can be reduce on this marine environment.

The inhibitory effect of Thymus vulgaris extracts on the planktonic form and biofilm structures of six human pathogenic bacteria.

OBJECTIVE: Microorganisms are responsible for many problems in industry and medicine because of biofilm formation. Therefore, this study was aimed to examine the effect of Thymus vulgaris (T. vulgaris) extracts on the planktonic form and biofilm structures of six pathogenic bacteria. MATERIALS AND METHODS: Antimicrobial activities of the plant extracts against the planktonic form of the bacteria were determined using the disc diffusion method. MIC and MBC values were evaluated using macrobroth dilution technique. Anti-biofilm effects were assessed by microtiter plate method. RESULTS: According to disc diffusion test (MIC and MBC), the ability of Thymus vulgaris (T. vulgaris ) extracts for inhibition of bacteria in planktonic form was confirmed. In dealing with biofilm structures, the inhibitory effect of the extracts was directly correlated to their concentration. Except for the inhibition of biofilm formation, efficacy of each extract was independent from type of solvent. CONCLUSION: According to the potential of Thymus vulgaris (T. vulgaris) extracts to inhibit the test bacteria in planktonic and biofilm form, it can be suggested that Thymus vulgaris (T. vulgaris) extracts can be applied as antimicrobial agents against the pathogenic bacteria particularly in biofilm forms.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Antibacterial Activity of Some Plant Extracts Against Extended- Spectrum Beta-Lactamase Producing Escherichia coli Isolates.

BACKGROUND: The extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli isolates make many serious infections, especially urinary tract infections. OBJECTIVES: The purpose of this study was to determine the antibacterial activities of some natural plant extracts against ESBL-producing E. coli isolates, which harbor the TEM gene in urine samples of the patients who have urinary tract infections. MATERIALS AND METHODS: Evaluation has to be exactly determined for both methods of disk diffusion test and polymerase chain reaction (PCR), separately. We evaluated 120 strains of E. coli isolates from the urine culture of the patients in Boo-Ali Hospital (Zahedan, south-eastern Iran) who were suffering from urinary tract infections. The ESBL-producing E. coli isolates were evaluated by disk diffusion test and PCR through TEM gene detection. The minimal inhibitory concentration (MIC) of commonly used antibiotics including ceftazidime, ceftriaxon, amikacin, gentamicin and ciprofloxacin along with the MIC of the alcoholic extract of different natural plants including Myrtus communis L (Myrtaceae), Amaranthus retraflexus (Amaranthaceae), Cyminum cuminum L (Apiaceae), Marrubium vulgare (Laminaceae) and Peganum. harmala (Zygrophyllaceae) against the ESBL-producing E. coli isolates, which harbor the TEM genes, were determined using the microdulition method. RESULTS: Results of this study showed that in disk diffusion method, 80 samples of E. coli produced ESBLs. In PCR method, the TEM gene distribution in the isolated ESBL-producing organisms was 50 (41.6%). Amikacin was the most effective anti-bacterial agent and ciprofloxacin was the least effective against E. coli isolates. All the natural plant extracts mentioned above, especially P. harmala, were effective against the selected isolates of ESBL-producing E. coli. The most frequent ESBL rate producing E. coli isolates (32 out of 50) had MIC of 2.5 mg/mL in ethanol extract of P. harmala. CONCLUSIONS: The alcoholic extract of P. harmala was very effective against the selected ESBL-producing E. coli isolates harboring the TEM gene. Therefore, it could be suggested as an antibacterial agent in the future. More researches are necessary for detecting the mechanism of this plant's behavior and its pharmacological effects.

Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance).

Biosurfactants are surface active materials that are produced by some microorganisms. These molecules increase biodegradation of insoluble pollutants. In this study sediments and seawater samples were collected from the coastline of Bushehr provenance in the Persian Gulf and their biosurfactant producing bacteria were isolated. Biosurfactant producing bacteria were isolated by using an enrichment method in Bushnell-Hass medium with diesel oil as the sole carbon source. Five screening tests were used for selection of Biosurfactant producing bacteria: hemolysis in blood agar, oil spreading, drop collapse, emulsification activity and Bacterial Adhesion to Hydrocarbon test (BATH). These bacteria were identified using biochemical and molecular methods. Eighty different colonies were isolated from the collected samples. The most biosurfactant producing isolates related to petrochemical plants of Khark Island. Fourteen biosurfactant producing bacteria were selected between these isolates and 7 isolates were screened as these were predominant producers that belong to Shewanella alga, Shewanella upenei, Vibrio furnissii, Gallaecimonas pentaromativorans, Brevibacterium epidermidis, Psychrobacter namhaensis and Pseudomonas fluorescens. The largest clear zone diameters in oil spreading were observed for G. pentaromativorans strain O15. Also, this strain has the best emulsification activity and reduction of surface tension, suggesting it is the best of thee isolated strains. The results of this study confirmed that there is high diversity of biosurfactant producing bacteria in marine ecosystem of Iran and by application of these bacteria in petrochemical waste water environmental problems can be assisted.

Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance).

Fifteen crude oil degrading bacteria were isolated from oil contaminated sites in the Persian Gulf at Khorramshahr provenance. These bacteria were screened with two important factors such as growth rate on crude oil and hydrocarbon biodegradation, and then three strains were selected from 15 isolated strains for further study. One strain (PG-Z) that show the best crude oil biodegradation was selected between all isolates. Nucleotides sequencing of the gene encoding for 16S rRNA show that strain PG-Z belong to Corynebacterium variabile genus. This strain was efficient in degrading of crude oil. This strain was capable to degraded 82% of crude-oil after one week incubation in ONR7a medium. The PG-Z strain had high emulsification activity and biosurfactant production between all isolates. GC-MS analysis shows that C. variabile strain PG-Z can degrade different alkanes in crude oil.

Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study.

Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a "Mesocosm Facility" (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2(T) (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2(T) and Thalassolituus oleivorans MIL-1(T) (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, ß-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2(T) alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable interaction between the two bacterial genera.

Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances).

Petroleum products spill and leakage have become two major environmental challenges in Iran. Sampling was performed in the petroleum reservoir waste water of Tehran and Kerman Provinces of Iran. Alkane degrading bacteria were isolated by enrichment in a Bushnel-Hass medium, with hexadecane as sole source of carbon and energy. The isolated strains were identified by amplification of 16S rDNA gene and sequencing. Specific primers were used for identification of alkane hydroxylase gene. Fifteen alkane degrading bacteria were isolated and 8 strains were selected as powerful degradative bacteria. These 8 strains relate to Rhodococcus jostii, Stenotrophomonas maltophilia, Achromobacter piechaudii, Tsukamurella tyrosinosolvens, Pseudomonas fluorescens, Rhodococcus erythropolis, Stenotrophomonas maltophilia, Pseudomonas aeruginosa genera. The optimum concentration of hexadecane that allowed high growth was 2.5%. Gas chromatography results show that all strains can degrade approximately half of hexadecane in one week of incubation. All of the strains have alkane hydroxylase gene which are important for biodegradation. As a result, this study indicates that there is a high diversity of degradative bacteria in petroleum reservoir waste water in Iran.

Effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics during assays of oil spill bioremediation: a microcosm study.

Microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics. An experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003), was monitored for 15 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out during all experimentation period. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil degradation was monitored by GC-MS analysis. Bioemulsificant addition stimulated an increase of the total bacterial abundance, change in the community structure and activity. The bioemulsificant also increased of 5 times the oil biodegradation rate. The data obtained from microcosm experiment indicated that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Isolation and characterization of two crude oil-degrading yeast strains, Yarrowia lipolytica PG-20 and PG-32, from the Persian Gulf.

Among six crude oil-degrading yeasts that were isolated from an oil-polluted area in the Persian Gulf, two yeast strains showed high degradation activity of aliphatic hydrocarbons. From an analysis of 18S rRNA sequences and biochemical characteristics, these strains were identified as Yarrowia lipolytica strains PG-20 and PG-32. Gas Chromatography (GC) analysis of the crude oil remaining in the culture medium after 1 week at 30°C showed that the strains PG-20 and PG-32 degraded 68% and 58% of crude oil, respectively. The optimal growth condition and biodegradation of hydrocarbons was in ONR medium with an acidic pH (pH5). These two strains may degrade aliphatic hydrocarbons more efficiently than aromatic hydrocarbons, although strain PG-20 had better degradation than strain PG-32. The two Y. lipolytica strains reduce surface tension when cultured on hydrocarbon substrates (1% v/v). These strains showed a cell surface hydrophobicity higher than 70%. These results suggested that Y. lipolytica strains PG-20 and PG-32 have high crude oil degrading activity due to their high emulsifying activity and cell hydrophobicity. In conclusion, these yeast strains can be useful for the bioremediation process in the Persian Gulf and decreasing oil pollution in this marine ecosystem.

Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea.

Twenty-five crude-oil-degrading bacteria were isolated from oil-contaminated sites in the Persian Gulf and the Caspian Sea. Based on a high growth rate on crude oil and on hydrocarbon degradation ability, 11 strains were selected from the 25 isolated strains for further study. Determination of the nucleotide sequence of the 16S rRNA gene showed that these isolated strains belonged to genera Acinetobacter, Pseudomonas, Gordonia, Rhodococcus, Cobetia, Halomonas, Alcanivorax, Marinobacter and Microbacterium. Among the 11 isolates, strains BS (Acinetobacter calcoaceticus, 98%) and PG-12 (Alcanivorax dieselolei, 98%) were the most effective in degrading crude oil. Rate of crude-oil degradation of 82% (isolate BS) and 71% (isolate PG-12) were observed after 1 week of cultivation in mineral medium. These strains had high emulsification activity and biosurfactant production. GC-MS analysis showed that A. dieselolei PG-12 can degrade different alkanes in crude oil. Screening of the distribution of the alkane hydroxylase gene in 25 isolates in relation to the source of isolation indicated that the group (II) alkane hydroxylase is prevalent in the Caspian Sea, but in the Persian Gulf, the frequency of the group (III) alkane hydroxylase gene is greater than that of the group (II) alkane hydroxylase gene.