AraC transcriptional regulator, aspartate semialdehyde dehydrogenase and acyltransferase: Three putative genes in phenol catabolic pathway of Acinetobacter sp. Strain DF4.

The objective of this study was to identify genes associated with the biodegradation of phenol by Acinetobacter sp. strain DF4 through the use of differential display (DD) methodology. The bacteria were grown in YEPG medium, and total RNA was extracted and analyzed using labeled primers to detect gene expression differences. Three distinctively expressed cDNA bands (ph1, ph2, and ph3) were identified, cloned, and sequenced. DNA analysis involved searching for open reading frames (ORFs), verifying results with the NCBI database, predicting promoter regions, and constructing phylogenetic trees using bioinformatics tools. The ph1 gene displayed a 97% identity with the AraC transcriptional regulator, suggesting its potential role in regulating the ortho-catabolic pathway of phenol. The ph2 gene showed a 98% identity with aspartate semialdehyde dehydrogenase, which is involved in phenol degradation. The ph3 gene had a 93% identity with acetyltransferase. Essential transcription factors, such as TATA, GTGTGT, CACA, and CTTTT, were detected, and the three genes promoter regions were predicted. This study successfully identified functional genes involved in the metabolism of cyclic chemicals, particularly phenol, using the DD technique. These findings provide insights into the biodegradation pathways of phenol by Acinetobacter sp. Strain DF4 and may contribute to the development of more efficient bioremediation strategies for phenol-contaminated environments.

Alpha-glucan: a novel bacterial polysaccharide and its application as a biosorbent for heavy metals.

This study identified an extracellular bacterial polysaccharide produced by Bacillus velezensis strain 40B that contains more than 90% of the monosaccharide glucose as alpha-glucan. A prominent peak at 1074 cm-1, a characteristic of glycoside couplings, was visible in the FTIR spectrum. There were traces of xylose, sucrose, and lactose, according to the HPLC study. The ability of this bacterial glucan to operate as a biosorbent of the heavy metals cobalt, chromium, copper, and lead from aqueous solutions was investigated in conjunction with Ca-alginate beads. It proved that glucan 40B has a low affinity for chromium ions and is selective for lead. Initial concentration measurements showed an inverse relationship between concentration and the amount of metal ions eliminated. Lead and chromium removal increased as the glucan dose was increased. It was shown that as the pH of the starting solution is elevated, there is an increase in the sorption of metal ions onto the glucan. It was clear that when the temperature increased, the fraction of metal ion sorption slightly increased. Glucan has a wide range of industrial applications, from food and medicine to health and nutrition. As a result, the investigation's scope was expanded to include heavy metal removal.

Back to nature: henna extracts from nanotech to environmental biotechnology - a review.

The Lythraceae family includes henna (Lawsonia inermis), which thrives in subtropical and tropical climates. One of its many and long-standing uses is in cosmetics as a pigment to color hair and nails. Additionally, it serves as a disinfectant against microbiological infections and has traditional applications in the textile industry, specifically for coloring wool and nylon. The dried leaves of henna contain a significant amount of lawsone, an active substance bestowing them with staining abilities. Environmental biotechnology, a subfield of biotechnology, engages in the production of biomass or renewable energy sources and the elimination of pollutants, utilizing either entire organisms or their by-products. Recent research indicates that henna, owing to its sustainability, abundant production, simplicity of preparation, low cost, and reputation for being safe and ecologically benign, is exceptionally well-suited to participate in the realm of environmental biotechnology. This review navigates through the most recent studies exploring the use of henna and its extracts for related purposes. These encompass a spectrum of applications, including but not limited to nanobiotechnology, fabric dyeing, corrosion resistance, colored solar cells, carbon dots, and new renewable energy exemplified by biofuel and biohydrogen. Furthermore, henna extracts have been deployed to function as antimicrobials and ward off dangerous insects.

Inhibitory Effects of Bacterial Silk-like Biopolymer on Herpes Simplex Virus Type 1, Adenovirus Type 7 and Hepatitis C Virus Infection.

Bacterial polymeric silk is produced by Bacillus sp. strain NE and is composed of two proteins, called fibroin and sericin, with several biomedical and biotechnological applications. In the current study and for the first time, the whole bacterial silk proteins were found capable of exerting antiviral effects against herpes simplex virus type-1 (HSV-1), adenovirus type 7 (AD7), and hepatitis C virus (HCV). The direct interaction between bacterial silk-like proteins and both HSV-1 and AD7 showed potent inhibitory activity against viral entry with IC50 values determined to be 4.1 and 46.4 µg/mL of protein, respectively. The adsorption inhibitory activity of the bacterial silk proteins showed a blocking activity against HSV-1 and AD7 with IC50 values determined to be 12.5 and 222.4 ± 1.0 µg/mL, respectively. However, the bacterial silk proteins exhibited an inhibitory effect on HSV-1 and AD7 replication inside infected cells with IC50 values of 9.8 and 109.3 µg/mL, respectively. All these results were confirmed by the ability of the bacterial silk proteins to inhibit viral polymerases of HSV-1 and AD7 with IC50 values of 164.1 and 11.8 µg/mL, respectively. Similarly, the inhibitory effect on HCV replication in peripheral blood monocytes (PBMCs) was determined to be 66.2% at concentrations of 100 µg/mL of the bacterial silk proteins. This antiviral activity against HCV was confirmed by the ability of the bacterial silk proteins to reduce the ROS generation inside the infected cells to be 50.6% instead of 87.9% inside untreated cells. The unique characteristics of the bacterial silk proteins such as production in large quantities via large-scale biofermenters, low costs of production, and sustainability of bacterial source offer insight into its use as a promising agent in fighting viral infection and combating viral outbreaks.

Unveiling the role of novel biogenic functionalized CuFe hybrid nanocomposites in boosting anticancer, antimicrobial and biosorption activities.

The quest for eco-friendly and biocompatible nanoparticles (NPs) is an urgent issue in the agenda of the scientific community and applied technology, which compressing synthesis routes. For the first time, a simple route for the biosynthesis of functionalized CuFe-hybrid nanocomposites (FCFNCs) was achieved using Streptomyces cyaneofuscatus through a simultaneous bioreduction strategy of Cu and Fe salts. The suitability of FCFNCs was evaluated medically and environmentally as an anticancer agent, antimicrobial agent and dye bio-sorbent. The physicochemical characteristics of FCFNCs using XRD, EDX, elemental mapping, FTIR, UV-Vis., TEM and ζ-potential confirmed the formation of spheres agglomerated into chains (37 ± 2.2 nm), self-functionalized nanocomposite by proteinaceous moieties with considerable stability (- 26.2 mV). As an anticancer agent, FCFNCs displayed the highest apoptotic impact (> 77.7%) on Caco-2, HepG-2, MCF-7 and PC-3 cancer cells at IC50 ≤ 17.21 µg/mL with the maximum up regulation of p53 and caspase 3 expression and the lowest Ki-67 level, relative to both functionalized CuNPs (FCNPs) and FeNPs (FFNPs). Meanwhile, it maintained the viability of normal human cells by EC100 up to 1999.7 µg/mL. Regarding the antimicrobial activity, FCFNCs offered > 70% growth reduction among wide spectrum prokaryotic and eukaryotic pathogens. Additionally, the synergistic feature of FCFNCs disintegrated the pre-established biofilm and algal growth in a dose-dependent manner. However, as a bio-sorbent, FCFNCs decolorized > 68% of malachite green and congo red dyes (200 mg/L), reflecting considerable remediation efficiency, confirmed by FTIR of FCFNCs- adsorbed dyes and microtoxicity/cytotoxicity of solutions after remediation. This study offers new insights into promising CuFe-hybrid nanocomposites for recruitment in several applications.

Aerobic and anaerobic removal of lead and mercury via calcium carbonate precipitation mediated by statistically optimized nitrate reductases.

The nonbiodegradability nature of heavy metals renders them resident in food chain and subsequently, destructing the entire ecosystem. Therefore, this study aimed to employ nitrate reduction-driven calcium carbonate precipitation in remediation of lead and mercury aerobically and anaerobically by Proteus mirabilis 10B, for the first time. Initially, Plackett-Burman design was employed to screen of 16 independent variables for their significances on periplasmic (NAP) and membrane-bound (NAR) nitrate reductases. The levels for five significant variables and their interaction effects were further optimized by central composite design. The maximum activities of NAP and NAR recorded 2450 and 3050 U/mL by 2-fold enhancement, comparing with non-optimized medium. Under aerobic and anaerobic optimized remediation conditions, the changes in media chemistry revealed positive correlation among bacterial growth, nitrate reductase activity, pH, NO3- and NO2- consumption and removal of Ca2+, Pb2+ and Hg2+. Subsequently, the remediated precipitates were subjected to mineralogical analysis; energy dispersive X-ray patterns exhibited characteristic peaks of C, O and Ca in addition to Pb and Hg. Scanning electron microscope depicted the presence of bacterial imprints and protrusions on rough and smooth surface bioliths. However, X-ray diffraction indicated entrapment of PbCO3, Pb2O, CaPbO3, Hg and Hg2O in calcite lattice. Interestingly, such approach is feasible, efficient, cost-effective and ecofriendly for heavy metals remediation.

In vitro assessment of the bioactivities of sericin protein extracted from a bacterial silk-like biopolymer.

Sericin is one of the main components of silk proteins, which has numerous biomedical applications because of its antioxidant, anticancer and antimicrobial properties. We recently isolated and characterized a novel silk-like protein named BNES. It is of non-animal origin and is like a bacterial polymeric silk. Sericin is a very popular protein compound that is effective in treating cancerous tumors. The process of extracting it from natural silk produced by silkworms or spiders is both complex and expensive. From the published scientific literature, it has been shown that sericin has not been previously extracted from a bacterial source. In the present study, sericin was extracted from bacteria capable of producing a biopolymer named BNES whose chemical composition is like that of natural silk and its bio-therapeutic effects were evaluated for the first time. The antioxidant activity of BNES measured by DPPH and ABTS assays showed IC50 values of 0.38 and 0.41 mg mL-1, respectively. BNES displayed satisfactory cytotoxic effect against four cancer cell lines, including Huh-7, Caco-2, MCF-7 and A549 cells, with IC50 values in the ranges of ca. 0.62 ± 0.17, 0.72 ± 0.27, 0.76 ± 0.36 and 0.83 ± 0.31 mg mL-1, respectively, after 24 h of treatment and 0.51 ± 0.22, 0.49 ± 0.19, 0.41 ± 0.25 and 0.55 ± 0.38, respectively, after 48 h of treatment, without affecting normal cells (WI38 cells). The antitumor activity of BNES was established to be an apoptosis-dependent mechanism determined via cellular morphology alterations, cell cycle arrest in the sub-G1 phase and nuclear staining with highly fluorescent fragments. The antimicrobial effects of BNES were examined with yeast and Gram-negative and Gram-positive bacteria. The results confirmed its antimicrobial activity against all tested organisms at concentrations of up to 1.33 mg mL-1. The competitive advantage of the bacterial sericin BNES over sericin extracted from spider or silkworm sources is that it can be produced in very large quantities through large-scale bio-fermenters, which reduces the expected cost of production, in addition to having sustainable bacterial production source.

Disinfection of water and wastewater by biosynthesized magnetite and zerovalent iron nanoparticles via NAP-NAR enzymes of Proteus mirabilis 10B.

Disinfection of water and wastewater strongly contributes to solving the problem of water shortage in arid/semi-arid areas; cheap and ecofriendly approaches have to be used to meet water quality standards. In the present study, a green synthesis of iron nanoparticles (INPs) under aerobic and anaerobic conditions via nitrate reductases (NAP/NAR) enzymes produced by Proteus mirabilis strain 10B were employed for this target. The biosynthesized INPs were characterized; UV-Vis spectroscopy revealed surface plasmon resonance at 410 (aerobic) and 265 nm (anaerobic). XRD indicated crystalline magnetite ((MNPs) aerobically synthesized) and zerovalent INPs (ZVINPs anaerobically synthesized). EDX demonstrated strong iron signal with atomic percentages 73.3% (MNPs) and 61.7% (ZVINPs). TEM micrographs illustrated tiny, spherical, periplasmic MNPs (1.44-1.92 nm) and cytoplasmic ZVINPs with 11.7-60.8 nm. Zeta potential recorded - 31.8 mV (ZVINPs) and - 66.4 mV (MNPs) affirming colloidal stability. Moreover, the disinfection power of INPs was evaluated for standards organisms and real water (fresh, sea and salt mine) and wastewater (municipal, agricultural and industrial) samples. The results reported that INPs displayed higher antagonistic effect than iron precursor, 700 and 850 µg/mL of MNPs and ZVINPs, respectively, was sufficient to show a drastic algicidal effect on algal growth. Both types of INPs demonstrated obvious dose-dependent antibiofilm efficiency. Due to their smaller size, MNPs were more efficient than ZVINPs at the suppression of microbial growth in all examined water samples. Overall, MNPs showed superior antagonistic activity, which promotes their exploitation in enhancing water/wastewater quality.

NAP enzyme recruitment in simultaneous bioremediation and nanoparticles synthesis.

The periplasmic nitrate reductase enzyme (NAP) has become attractive catalyst, whose exploitation has emerged as one of the indispensable strategies toward environmentally benign applications. To achieve them efficiently and overcome the sensitivity of NAP in harsh environmental circumstances, the immobilization for denitrifying bacteria and NAP enzyme for simultaneous bioremediation and bionanoparticles synthesis was studied. NAP catalyzed NO3- reduction at Vmax of 0.811 µM/min and Km of 14.02 mM. Concurrently, the immobilized MMT cells completely removed NO3- upon 192 h with AgNPs synthesis ranging from 23.26 to 58.14 nm as indicated by SEM. Wherase, immobilized NAP exhibited lower efficiency with 28.6% of NO3- elimination within 288 h and large aggregated AgNPs ranging from 94.44 nm to 172.22 nm. To the best of author knowledge, the immobilization for denitrifying bacteria and NAP enzyme for simultaneous bioremediation and bionanoparticles synthesis was not studied before.

Isolation, characterization, optimization, immobilization and batch fermentation of bioflocculant produced by Bacillus aryabhattai strain PSK1.

Among others, isolate PSK1 was selected and identified by 16 S rDNA sequencing as Bacillus aryabhattai. Growth optimization of PSK1 and physicochemical parameters affected bioflocculant production was carried out by Plackett-Burman design and resulted in increasing in the activity by 4.5%. Bioflocculant production by entrapped and adsorbed immobilized microbial cells was performed using different techniques and revealed enhancement in the activity in particular with pumice adsorption. HPLC analysis of sugars and amino acids composition, FTIR and the effect of different factors on the purified PSK1 biopolymer such as presence of cations, thermal stability, pH range and clay concentration was carried out. Scanning electron microscopy (SEM) of free, immobilized cells, PSK1 bioflocculant and formed flocs were performed. The results revealed that bioflocculant PSK1 is mainly glycoprotein consists of glucose and rhamnose with a large number of amino acids in which arginine and phenylalanine were the major. SEM analysis demonstrated that PSK1 have a clear crystalline rod shaped structure. FTIR spectrum reported the presence of hydroxyl and amino groups which are preferred in flocculation process. PSK1 was soluble in water and insoluble in all other tested organic solvents, while it was thermally stable from 40 to 80 °C. Among examined cations, CaCl2 was the best coagulant. The maximum flocculation activity of the PSK1 recorded at 50 °C (92.8%), pH 2.0 (94.56%) with clay concentration range 5-9 g/l. To obtain a large amount of PSK1 bioflocculant with high flocculating activity, batch fermentation was employed. The results recorded ∼6 g/l yield after 24 h of fermentation.

Characterization and flocculation properties of a carbohydrate bioflocculant from a newly isolated Bacillus velezensis 40B.

In this study, a bioflocculant with a high flocculation activity (> 98%) produced by strain 40B, which was isolated from a brackish water was investigated By 16S rDNA sequence analysis, strain 40B was identified as Bacillus velezensis. Chemical analysis of the bioflocculant 40B indicated that it contained 2% protein and 98% carbohydrates. FTIR analysis showed the presence of carboxyl, hydroxyl and amino groups, which were preferred for the flocculation process. The optimal concentration for the flocculation activity was 3.5 mg (-1). This polysaccharide could also flocculate kaolin suspension over a wide range of pH (1-10) and temperature (5-85 degrees C) in the presence of CaCl2. The stability of the bioflocculant 40B under various conditions suggests its possible use in the industries and environmental applications. However, no previous report exists on the isolation and characterization of a bioflocculant from the Bacillus velezensis.

Bacillus mojavensis strain 32A, a bioflocculant-producing bacterium isolated from an Egyptian salt production pond.

Bacillus mojavensis strain 32A that exhibited 96.11% flocculation efficiency for clay suspensions was selected from other 15 comparative strains. Under growth condition, strain 32A was able to produce 5.2g/L of purified biopolymer. Its constituent was mainly polysaccharide and protein with proportional of 98.4-1.6% respectively. FTIR spectrum was confirming its chemical analysis. This biopolymer attain very fast sedimentation rate. The cost-effective biopolymer and CaCl(2) dosages were 3mg/L and 5 ml/L respectively that posed 89.7% flocculation efficiency. These dosages were suitable only for clay concentrations ≤5g/L. The maximum flocculation efficiency of the biopolymer recorded at pH 1.0 of clay suspension. The too high (>75°C) or too low (<25°C) clay suspension temperature was unfavorable for the biopolymer flocculation performance. The biopolymer solution utilized high thermal stability over the temperature range of 5-60°C. Furthermore, its pH stability recorded at pH range of 5-9.

Biosynthesis of polyhydroxyalkanotes in wild type yeasts.

Biosynthesis of biodegradable polymers polyhydroxyalkanotes (PHAs) have been studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHAs in wild type yeasts is not well documented. The purpose of this study was to screen yeast isolates collected from different ecosystems for their ability to accumulate PHAs. Identification of the isolates and characterization of PHAs produced by the positive isolates were investigated. One positive isolate (strain Y4) was identified by both API20C system and 18S rDNA sequencing. The data revealed that isolate Y4 belongs to the yeast genus Rhodotorula and exhibits 18S rDNA similarity value >99% to the species Rhodotorula minuta. Quantification of PHAs yield of strain Y4 in glucose, oleic acid and tween 60 containing medium for over a growth period of 96 h gave 2% of PHAs in biomass. The nature of produced PHAs was analyzed by infrared spectroscopy and nuclear magnetic resonance (1H and 13C NMR) and found to contain polyhydroxybutyrate and polyhydroxyvalerate (PHBV).

Isolation and characterization of extracellular bioflocculants produced by bacteria isolated from Qatari ecosystems.

Compared with conventional synthetic flocculants, bioflocculants has special advantages such as safety, strong effect, biodegradable and harmlessness to humans and the environment, so they may potentially be applied in drinking and wastewater treatment, downstream processing, and fermentation processes. To utilize bioflocculants widely in industrial fields, it is desirable to find various microorganisms with high bioflocculant-producing ability and improve the flocculating efficiency of the bioflocculant. In the present study, screening of new flocculant-producing microorganisms was carried out using samples collected from different Qatari ecosystems. The flocculating activity of the novel bioflocculants produced by isolated microorganisms was investigated. A total of 5 g/l Kaolin suspension was used to measure the flocculating activity. Isolated bioflocculant-producing bacteria were identified by 16S rDNA analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (approximately 550 bp) in the GenBank database revealed that these bacteria are related to the genus Bacillus. FT-IR spectrometry analysis of the extracted bioflocculants indicated the presence of carboxyl, hydroxyl and amino groups preferred for the flocculation process. Influences of pH and bioflocculant dosage on the flocculation were also examined. The maximum flocculating rates were observed at pH 7, 7 and 3 of the bioflocculants derived from strains QUST2, QUST6 and QUST9, respectively. However, 20.0 mg/l was the dose that gave the highest flocculating rate with all examined bioflocculants. The elemental analysis of examined bioflocculants revealed the mass proportion of C, H, N and S. Carbon and nitrogen contents of examined bioflocculants were in the range of 42-48% and 11-12%, respectively.

Influence of phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter bacterium.

In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 degrees C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes.

Detection of nitrate/nitrite bioavailability in wastewater using a luxCDABE-based Klebsiella oxytoca bioluminescent bioreporter.

In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate (R2 = 0.98), 1 to 6 ppm for nitrite (R2 = 0.99), and 1 to 7 ppm for ammonium (R2 = 0.99). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.

Acinetobacter bioreporter assessing heavy metals toxicity.

This work was conducted to employ a whole cell-based biosensor to monitor toxicity of heavy metals in water and wastewater. An isolate of industrial wastewater bacterium, Acinetobacter sp. DF4, was genetically modified with lux reporter gene to create a novel bioluminescent bacterial strain, designated as DF4/PUTK2. This bioreporter can investigate the toxicity through light inhibition due to cell death or metabolic burden and the specific stress effects of the tested soluble materials simultaneously. The use of Acinetobacter DF4/PUTK2 as a bioluminescent reporter for heavy metal toxicity testing and for the application of wastewater treatment influent toxicity screening is presented in this study. Among eight heavy metals tested, the bioluminescence of DF4/PUTK2 was most sensitive to Zn, Cd, Fe, Co, Cr followed by Cu in order of decreasing sensitivity. The same pattern of sensitivity was observed when several contaminated water and wastewater effluents were assayed. This work suggested that luxCDABE -marked Acinetobacter bacterium DF4/PUTK2 can be used to bioassay the ecotoxicity of wastewater and effluent samples contaminated with heavy metals. Using this assay, it is possible to pre-select the more toxic samples for further chemical analysis and to discard wastewater samples with low or no inhibition because they are not toxic to the environment.

Genotypic discrimination between a Leishmania isolate and two Leishmania major reference strains using PCR-RFLP technique.

The restriction fragment length polymorphism (PCR-RFLP) of 18S rDNA amplified fragments, was conducted to recognize between an unidentified Leishmania isolated from Egyptian patient infected in Saudi Arabia and two L. major reference strains causing cutaneous lesions. The strains were maintained both in vivo & in vitro. Additional requirement as morphological characterization on basis of the light microscope & scanning electron microscopy and behaviour in experimental Swiss albino mice regarding development of lesions were performed. The results showed that, PCR-RFLP analysis of the 18S rDNA amplified PCR fragments was highly successful to put the unidentified Leishmania strain in the category of L. major. There was no significant differences regarding the cutaneous lesions development. In spite of the significant variations of the morphometric measurements of the three strains were observed.

Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria.

In this work we developed and optimized two molecular-based approaches to monitor rapidly, sensitively and specifically bacterial pathogens from three different genera, Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., directly in waters. To achieve this aim, firstly a multiplex-PCR assay (M-PCR) was optimized using a primer pair specific for each pathogen. Secondly, as a molecular confirmatory test after isolation of the pathogens by classical microbiological methods, PCR-RFLP of their amplified 16S rDNA genes was performed. It was observed from the results that the developed M-PCR assay has significant impact on the ability to detect sensitively, rapidly and specifically the three pathogens directly in water within a short time (5 h from sampling to obtain final results), therefore it represents a considerable advancement over other known more time-consuming and less-sensitive methods for identification and characterization of these kinds of pathogens.

Long PCR-amplified rDNA for PCR-RFLP- and Rep-PCR-based approaches to recognize closely related microbial species.

Long PCR was used to amplify a 5-kb fragment of the bacterial ribosomal operon (16S-intergenic spacer region (ISR)-23S) from several Ralstonia eutropha strains (16S rDNA sequence similarity: 97-99%). Due to the large product size, amplicons from the different strains could be distinguished using restriction enzyme fragment length polymorphisms (RFLP) and repetitive PCR analysis (Rep-PCR) with the primer 1492r. These methods may prove useful in differentiating other bacterial strains with highly similar 16S rDNA sequences.