Nitrate removal from industrial wastewater using six newly isolated strains of aerobic heterotrophic denitrifiers in an attached growth.

BACKGROUND: The objective of this study was to isolate specific heterotrophic aerobic denitrifying bacteria from a wastewater treatment plant and employ them in an attached growth system for wastewater denitrification. METHODS: To isolate and screen aerobic denitrifiers, Denitrifying Medium (DM) and Screen Medium (GN) were utilized. The Polymerase Chain Reaction (PCR) technique and 16S rDNA sequencing were used to identify the isolates. The formation of biofilms by selected isolates on ceramic media was examined using a Scanning Electron Microscope (SEM). This study also investigated various variables for nitrate removal, including temperature, Carbon/Nitrogen ratio (C/N), and the carbon source. A series of experiments were conducted to gauge nitrate removal under optimal variable values. RESULTS: Six purified strains exhibited the highest denitrification efficiency in less than 30 h. Pseudomonas species were chosen for additional experiments. Denitrification efficiencies ranged from a low of 71.4% (at a temperature of 30 °C, C/N ratio of 17, and citrate as the carbon source) to a high of 98.9% (at a temperature of 33 °C, C/N ratio of 8, and citrate as the carbon source). The average denitrification efficiency was 84.02%. Optimal nitrate removal occurred at temperatures around 30-31 °C and C/N ratios of approximately 5.8-6.5. CONCLUSION: This study demonstrates that aerobic denitrifying bacteria can effectively remove nitrate from aqueous solutions.

Efficacious antibacterial potency of novel bacteriophages against ESBL-producing Klebsiella pneumoniae isolated from burn wound infections.

BACKGROUND AND OBJECTIVES: Prevalence of extended spectrum ß-lactamase (ESBL) leads to the development of antibiotic resistance and mortality in burn patients. One of the alternative strategies for controlling ESBL bacterial infections is clinical trials of bacteriophage therapy. The aim of this study was to isolate and characterize specific bacteriophages against ESBL-producing Klebsiella pneumoniae in patients with burn ulcers. MATERIALS AND METHODS: Clinical samples were isolated from the hospitalized patient in burn medical centers, Iran. Biochemical screenings and 16S rRNA gene sequencing were determined. The phages were isolated from municipal sewerage treatment plants, Isfahan, Iran. TEM and FESEM, adsorption velocity, growth curve, host range, and the viability of the phage particles as well as proteomics and enzyme digestion patterns were examined. RESULTS: The results showed that Klebsiella pneumoniae Iaufa_lad2 (GenBank accession number: MW836954) was confirmed as an ESBL-producing strain using combined disk method. This bacterium showed significant sensitivity to three phages including PɸBw-Kp1, PɸBw-Kp2, and PɸBw-Kp3. Morphological characterization demonstrated that the phage PɸBw-Kp3 to the Siphoviridae family (lambda-like phages) and both phages PɸBw-Kp1 and ɸBw-Kp2 to the Podoviridae family (T1-like phages). The isolated bacteriophages had a large burst size, thermal and pH viability and efficient adsorption rate to the host cells. CONCLUSION: In present study, the efficacy of bacteriophages against ESBL pathogenic bacterium promises a remarkable achievement for phage therapy. It seems that, these isolated bacteriophages, in the form of phage cocktails, had a strong antibacterial impacts and a broad-spectrum strategy against ESBL-producing Klebsiella pneumoniae isolated from burn ulcers.

Bacteriophages PɸEn-CL and PɸEn-HO can eliminate MDR Enterobacter cloacae and Enterobacter hormaechei isolated from burn wound infections without toxicity for human skin cells.

The prevalence of multidrug-resistant (MDR) strains has caused serious problems in the treatment of burn infections. MDR Enterobactercloacae and Enterobacterhormaechei have been defined as the causative agents of nosocomial infections in burn patients. In this situation, examination of phages side effects on human cell lines before any investigation on human or animal that can provide beneficial information about the safety of isolated phages. The aim of this study was to isolate and identify the specific bacteriophages on MDR E. cloacae and E. hormaechei isolated from burn wounds and to analyze the efficacy, cell viability and cell cytotoxicity of phages on A-375 and HFSF-PI cell lines by MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) colorimetric assay and lactate dehydrogenase (LDH) release assay. Phages were isolated from urban sewage Isfahan, Iran. Enterobactercloacae strain Iau-EC100 (GenBank accession number: MZ314381) and E. hormaechei strain Iau-EHO100 (GenBank accession number: MZ348826) were sensitive to the isolated phages. Transmission electron microscopy (TEM) results revealed that PɸEn-CL and PɸEn-HO that were described had the morphologies of Myovirus and Inovirus, respectively. Overall, MTT and LDH assays showed moderate to excellent correlation in the evaluation of cytotoxicity of isolated phages. The results of MTT and LDH assays showed that, phages PɸEn-CL and PɸEn-HO had no significant toxicity effect on A375 and HFSF-PI 3 cells. Phage PɸEn-HO had a better efficacy on the two tested cell lines than other phage. Our results indicated that, there were significant differences between the two cytotoxicity assays in phage treatment compared to control.

Isolation, characterization, and effectiveness of bacteriophage Pɸ-Bw-Ab against XDR Acinetobacter baumannii isolated from nosocomial burn wound infection.

OBJECTIVES: With emergence of drug resistance, novel approaches such as phage therapy for treatment of bacterial infections have received significant attention. The purpose of this study was to isolate and identify effective bacteriophages on extremely drug-resistant (XDR) bacteria isolated from burn wounds. MATERIALS AND METHODS: Pathogenic bacteria were isolated from hospitalized patient wounds in specialized burn hospitals in Iran, and their identification was performed based on biochemical testing and sequencing of the gene encoding 16S rRNA. Bacteriophages were isolated from municipal sewage, Isfahan, Iran. The phage morphology was observed by TEM. After detection of the host range, adsorption rate, and one-step growth curve, the phage proteomics pattern and restriction enzyme digestion pattern were analyzed. RESULTS: All isolates of bacteria were highly resistant to antibiotics. Among isolates, Acinetobacter baumannii strain IAU_FAL101 (GenBank accession number: MW845680), which was an XDR bacterium, showed significant sensitivity to phage Pɸ-Bw-Ab. TEM determined the phage belongs to Siphoviridae. They had double-stranded DNA. This phage showed the highest antibacterial effect at 15 °C and pH 7. Analysis of the restriction enzyme digestion pattern showed Pɸ-Bw-Ab phage was sensitive to most of the used enzymes and based on SDS-PAGE, protein profiles were revealed 43 to 90 kDa. CONCLUSION: Considering the potential ability of the isolated phage, it had an antibacterial impact on other used bacterial spp and also strong antibacterial effects on XDR A. baumannii. Also, it had long latency and low burst size. This phage can be a suitable candidate for phage therapy.

Production of glycolipid biosurfactant during crude oil degradation by the novel indigenous isolated Achromobacter kerstersii LMG3441.

This study aimed to find biosurfactant producing and crude oil-degrading bacteria able to decontaminate crude oil from wastewater. The bacteria that were isolated from contaminated sites in an oil refinery plant in Isfahan, Iran, were identified by 16S rDNA sequencing as Achromobacter kerstersii strain LMG3441, Klebsiella pneumonia strain SKBA6, and Klebsiella variicola strain SKV2. According to the results obtained from different tests for the production of biosurfactant among three strains, only Achromobacter kerstersii strain LMG3441 was selected for further study. The pattern of residual hydrocarbons was analyzed by high-resolution gas chromatography-mass spectrometry (GC-MS). This novel and indigenous strain was capable of producing the highest amount of a glycolipid biosurfactant (7.81 g/L) in MSM (mineral salt medium) with 1% (v/v) crude oil as the only source of carbon and energy. The compound showed high surface activation capacity with reduction of surface tension from 40 mN m-1 in the control to 23.3 mN m-1 by the bacterium. The results of GC-MS for assessment of residual hydrocarbons in the MSM and comparison with crude oil as a control showed that 53% of the hydrocarbons in the crude oil were consumed by this novel strain.

The Effect of Environmental Stresses on lipL32 Gene Expression in Pathogenic Leptospira spp. through Real-Time PCR.

Leptospirosis is a worldwide infectious and zoonotic disease. The incidence of this disease is high in temperate regions, especially in northern Iran. The aim of this study was to investigate the effects of temperature, pH, and Phyllanthus amarus plant extract on the lipL32 gene expression in pathogenic Leptospira spp. Fifty water samples were collected. Culture and PCR technique were used to isolate and identify the bacterium and the presence of the lipL32 gene. The samples were exposed to different temperatures and pH levels for one day and the Ph. amarus plant extract at different concentrations for one and seven days. RNA was extracted, and cDNA synthesis was performed for all the samples. All cDNAs were evaluated by the real-time PCR (SYBR green) technique. Out of the 50 samples, ten samples (20%), using PCR were determined to contain the pathogenic Leptospira. Fold change of the expression of the lipL32 gene associated with stresses was as follows: temperature stress of 40°C, 35°C, and 25°C reduced the lipL32 gene expression in all three isolates, especially in the isolates type 1. The pH stress, i.e., pH values equal to 8 or 9 reduced the gene expression in three types of isolates, and pH = 6 stress increases the lipL32 gene expression in the isolates of type 1. Ph. amarus plant extract stress reduced the mentioned gene expression only in isolates of type 2. Temperature and pH stresses could lead to differences in the expression level and cause the lipL32 gene expression decrease in three pathogenic isolates. The MIC results showed anti-leptospiral effect of Ph. amarus plant extract.

Using autochthonous Bdellovibrio as a predatory bacterium for reduction of Gram-negative pathogenic bacteria in urban wastewater and reuse it.

BACKGROUND AND OBJECTIVES: The microbial contamination of wastewater is associated with health risks. The aim of this study was to use the autochthonous Bdellovibrio potential to prey Gram-negative pathogenic bacteria as a bio-control agent to treat urban wastewater. MATERIALS AND METHODS: Thirty-six raw sewage samples were collected for isolation of Bdellovibrio. Double layer plaque assay was used for isolation and the isolates were identified by microscopic examination and molecular analysis. To evaluate the predatory potential for decrease number of Gram-negative pathogenic bacteria, plaque perdition assay, reduction in host cells viability by colony-forming unit (CFU) counting, reduction in optical density (OD) in co-cultures and assay of killing efficiency were carried out. Also, the raw wastewater was treated by Bdellovibrio then the reduction in CFU counting and reduction in OD was evaluated. RESULTS: Four strains of Bdellovibrio were isolated and were registered in Gene Bank. Clear plaques were observed after 3-6 days of incubation for all prey cells. The CFU enumerations of all preys were decreased after 48 hrs in co-cultures and raw wastewater. Also, OD was decreased down to 0.2 nm after 48 hrs. CONCLUSION: These autochthonous Bdellovibrio strains are proposed to use for bio-control of Gram-negative pathogenic bacteria in wastewater and reuse it for irrigation in arid regions.