Molecular investigation of CTX-M gene in Extended Spectrum β Lactamases (ESBLs) producing Pseudomonas aeruginosa isolated from Iranian patients with burn wound infection

Background: Pseudomonas aeruginosa (P. aeruginosa) is one of the most important causes of infection in burns and intensive care units. Extended-spectrum β-lactamases (ESBLs) production in P. aeruginosa is a major factor in the antibiotic resistance and is thought of as a serious threat to the currently available antibiotic armory. The purpose of this study was to determine the prevalence of CTX-M gene in ESBL-producing P. aeruginosa isolates in burn wound samples.Materials and methods: In this cross-sectional survey, a total of 60 clinical isolates of P. aeruginosa were obtained from patients suffering from burn wound infection referred to major hospitals of Tehran, Iran. After verification by biochemical tests and antimicrobial susceptibility testing, CTX-M gene was identified using PCR method.Results: The results of the molecular analysis of CTX-M gene showed that the prevalence of isolates of P. aeruginosa harboring CTX-M gene was 20% (12/60).Conclusion: The results from this study showed high levels of antibiotic resistance and CTX-M gene among P. aeruginosa isolated samples of burn-wound infections which condition may result in the increased the emergence of multidrug-resistant strains and the failure of therapy This study suggests that detailed data on the CTX-M gene frequency can be useful to achieve the best therapy for infections caused by ESBLs producing P. aeruginosa

Growth Optimization of Lactobacillus plantarum T5jq301796.1, an Iranian Indigenous Probiotic in Lab Scale Fermenter

Background and Objective: Lactobacillus plantarum is one of the probiotics species used in functional food products. These bacteria or their purified bacteriocins are used as biological preservatives in the food industry. The first step in production of an array of probiotic products is optimizing production in fermentors. This study aimed to examine factors affecting the in vitro growth optimization of Lactobacillus plantarum T5JQ301796.1 in a lab scale fermentor.Materials and Methods: Following 24 hours of anaerobic culture of the lactobacillus at 37°C, the pre-culture was ready and was inoculated to a 5 liter fermentor at 37°C and stirred at 40 rpm. Then factors affecting lactobacillus growth including carbon and nitrogen sources and pH were studied. The results were interpreted using response surface methodology (RSM), and optimal conditions for the equipment were determined.Results and Conclusion: For optimal growth of Lactobacillus plantarum T5JQ301796.1 in lab scale fermentor, the optimal conditions were 25.96 gl-1 of glucose, 1.82% of yeast extract, pH of 7.26, and stirring at 40 rpm at optimum temperature between 37-40°C. In this condition, maximum viable cell in the batch fermentation was 1.25×1010 CFU ml-1. Application of central composite design for the growth optimization of this bacterium led to maximum viable cells equal to 1.25×1010 CFU ml-1. So the mentioned features can lead to optimum industrial scale production and usage of this probiotic strain in probiotic products.Conflict of interest: The authors declare that there is no conflict of interest

Characterization of Effective Native Lactic Acid Bacteria as Potential Oral Probiotics on Growth Inhibition of Streptococcus mutans

Background and Objective: Probiotics' effects on harmful oral bacteria have been verifed. As antibiotic resistance becomes a major problem, searching for novel potential species is important. The objective of this study was to select novel safe strains of lactic acid bacteria with potentials as oral probiotics. Furthermore, ability of these strains to suppress growth and attachment of Streptococcus mutans as the most important cariogenic bacteria in tooth decay was investigated. Material and Methods: Initial identification tests, including Gram staining and catalase and oxidase tests, were carried out on 22 strains of lactic acid bacteria isolated from Iranian traditional dairy products. Safety of the strains was assessed using hemolysis test and antibiotic resistance assessment. Strains were then assessed for probiotic characteristics such inhibition of Streptococcus mutans growth, tolerance to lysozyme enzymes and ability of adhesion as well as ability of decreasing Streptococcus mutans adhesion. Selected strains were identified using16S rRNA molecular method. Results and Conclusion: Of all strains, four strains with the optimal probiotic characteristics were selected. These included one Lactobacillus brevis, one Lactobacillus casei and two Lactobacillus paraceasei. These four strains showed strong antimicrobial characteristics against Streptococcus mutans, were resistant to oral lysozyme enzymes and included high adhesion abilities to polystyrene wells. Furthermore, they decreased Streptococcus mutans attachment; thus, biofilm formation by this bacterium was prevented. These strains were recognized as safe strains since they were approved in assessments of antibiotic susceptibility and hemolytic activity. Therefore, these four strains are suggested as oral probiotics. Conflict of interest: The authors declare no conflict of interest

Production and Characterization of Biosurfactants Using Bacteria Isolated from Acidic Hot Springs

  Background and objective: Biosurfactants are increasingly used by food industries due to their low toxicities and unique structures. In this study, biosurfactants were produced and characterized for the first time using acidic bacteria isolated from acidic hot springs in Bushehr Province, Iran. Material and methods: Screening and identification of the most efficient species for biosurfactant production were carried out on 12 bacterial species using several experiments such as hemolysis, surface tension, emulsification index and diameter of clear zone. In addition to biosurfactant production, kinetics, stability and structural and thermal analysis were carried out for the bacterial strains using thin layer chromatography, Fourier Transform Infrared, nuclear magnetic resonance and differential scanning calorimetry. Results and conclusion: The biosurfactant from the selected bacteria (0.1 g l-1) was thermally stable at 120°C for 30 min. Stability at temperatures up to 140°C was confirmed using differential scanning calorimetry. The most significant novelty included the fact that the surface property was preserved until an osmolarity of 4% w v-1. Decreased surface tension and the emulsification potential were only reported at concentrations higher the highlighted concentration. Biological assay showed that Staphylococcus aureus was susceptible to produced biosurfactants, while no susceptibility was seen in Escherichia coli. Degeneration of SW480 cell line exposed to 0.601 µg µl-1 of the biosurfactant was detected after 24 h. The structural analysis showed that the biosurfactant was similar to surfactin as a food bioemulsifier. Conflict of interest: The authors declare that they have no conflict of interest

Effect of Ethanolic and Aqueous Extracts of Purslane on Probiotic Bacteria (Lactobacillus acidophilus and Lactobacillus casei)

ABSTRACT The use of natural ingredients such as herbs had a tremendous growth in food preservation. These compounds, in addition to antimicrobial properties, have flavoring and antioxidant properties as well. Recent research suggests that certain live microorganisms may play a role in regulating the immune system and cancer prevention that of among them to the probiotic bacteria can be noted. On the other hand, probiotic products by improving the intestinal microbial flora and digestive tract, and boost the immune system have significant impact on consumers' health. This study was conducted to evaluate the effect of aqueous and ethanolic extracts of Purslane on activities of probiotic bacteria Lactobacillus casei strain T4 and Lactobacillus acidophilus strain LA-5. The purpose of this study was to evaluate the effect of Purslane on the growth of Lactobacillus bacteria hence was prepared the aqueous and ethanolic extracts of Purslane and probiotic bacteria were exposed to various concentrations (2.5, 5 and 7%)of aqueous and ethanolic extracts. The results were evaluated after 24 hours by spectrophotometry and optical density (OD) methods. The results showed that some concentrations (2.5 and 5 %) of ethanol extract of Purslane and only the 7% concentration of aqueous extracts of purslaneincrease the growth of LA-5, whereas, the aqueous extract and also ethanolic extract of purslane at certain concentrations, can reduce the growth of T4

DNA Fingerprinting Based on Repetitive Sequences of Iranian Indigenous Lactobacilli Species by (GTG)5- REP-PCR

Background and Objective: The use of lactobacilli as probiotics requires the application of accurate and reliable methods for the detection and identification of bacteria at the strain level. Repetitive sequence-based polymerase chain reaction (rep-PCR), a DNA fingerprinting technique, has been successfully used as a powerful molecular typing method to determine taxonomic and phylogenetic relationships among bacteria. The aim of this study was to evaluate and detect the genetic diversity of lactobacilli species isolated from different sources in Iran. Material and Methods: Twenty strains were isolated from Iranian traditional yoghurt, cheese, and Tarkhineh. PCR-mediated amplification was carried out by degenerate primers. Sequencing was performed after purification of the PCR product. The rep-PCR fingerprinting by (GTG) 5 oligonucleotide primers was conducted for the discrimination and genotypic grouping of isolates. Results: Isolates were deposited as novel stains of lactobacillus casei, brevis, plantarum, and Entrococcus facium in GenBank. Clustering methods were performed on molecular data by NTSYS software, which was also supported by PCO ordination plot. The rep-PCR profiles showed that the 20 isolates produced different banding patterns. In UPGMA dendrogram, three main clusters were formed. Conclusion: According to our findings, rep-PCR appeared to be a very practical method and highly sensitive in the discrimination of the lactobacillus species. The results of sequencing corresponded to the clustering in dendrogram

Genotypic and Phylogenic Analysis of Lactobacilli Producing Bacteriocin Isolated from Traditional Dairy Products and Food

Background & Objective: Lactic acid bacteria (LAB) are a group of Gram-positive, non-spore forming, cocci or rod shaped, catalase negative organisms, considered as Generally Recognized as Safe (GRAS) organisms. These bacteria are used for thousands of years for production of fermented foods because of their ability to produce desirable changes in taste, flavor and texture. Different antimicrobial molecules such as bacteriocins produced by these bacteria that can inhibit food pathogens, so enhancing the shelf life and improving the safety of food products. Because of important role of LAB to improving the human health, molecular identification and phylogenic analysis of these bacteria based on 16S rRNA sequencing play the critical role in investigation of local sources of LAB in Iran. Materials & Methods: 5 isolates were selected from 20 isolates for molecular identification. These strains produced the high level of bacteriocin. Total genomic DNA was extracted by lysosyme extraction protocol. PCR-mediated amplification was carried out by degenerate primers. Sequencing was performed after purification of PCR product. Results: Isolates were deposited as novel strains of Lactobacillus casei and Entrococcus facium in GenBank. Conclusion: Because of high potential of local probiotic bacteria in Iran, these strains may be useful and could be used in the food industry

Molecular detection of heat-killed probiotic bacteria and study of apoptosis induction on colon cancer HT-29 cell line

Background and Aim: Due to the high prevalence of colon cancer in human societies such as Iran, the aim of this study is the molecular identification and assesses cytotoxic effects of heat-killed probiotic bacteria and induced apoptosis in HT-29 cell lines of colon cancer compared with normal cells. Materials and Methods: Molecular detection was performed on local probiotic bacteria isolated (TD4) from Tarkhineh dough. Both cell lines HT-29 colon cancer and HEK-293 were affected by 0.01, 0.1, 1, 10, 100, and 1000 µg/ml of heat-killed TD4 isolate for periods of 24, 48, and 72 hours. Cell viability was measured using MTT assay. DNA fragmentation assay was performed after 48 and 72 hours base on the IC50 concentration of heat-killed bacteria. Results: TD4 isolate was submitteded lactobacillus berevis in GenBank. According to MTT assay, lactobacillus berevis depended on time and dose reduced survival and proliferation of colon cancer of HT-29 cell line, with the highest cytotoxic effect in 1000 µg/ml for 72 hours. At this dose and time, viability was 23% and 50% in HT-29 and HEK-239 cell lines, respectively. DNA fragmentation assay showed apoptosis induction by heat-killed bacteria in the HT-29 cell line. Conclusions: L.berevis showed cytotoxic effects and induced apoptosis on HT-29 colon cancer cell line while the cytotoxic effect was slight on HEK-239 compared to HT-29. Future studies are still required to confirm these bacteria as a biological anti-cancer product in treatment and prevention

Histological and bacteriological changes in intestine of beluga (Huso huso) following ex vivo exposure to bacterial strains

In the present study the intestinal sac method (ex vivo) was used to evaluate the interactions between lactic acid bacteria and staphylococci in the gastrointestinal (GI) tract of beluga (Huso huso). The distal intestine (DI) of beluga was exposed ex vivo to Staphylococcus aureus, Leuconostoc mesenteroides and Lactobacillus plantarum. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples and samples exposed only to Leu. mesenteroides and a combination of Leu. mesenteroides and Staph. aureus, had a similar appearance to intact intestinal mucosal epithelium, with no signs of cellular damage. However, exposure of the DI to Staph. aureus and L. plantarum resulted in damaged epithelial cells and disorganized microvilli. Furthermore, 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the adherent microbiota of distal beluga intestine. Several bacterial species were identified by DGGE in the present study that have not previously been identified in beluga