Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)

It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log₁₀ c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log₁₀ c.f.u./ml decrease in biofilm formation and a 1.5 log₁₀ c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.     

Antibacterial activities of nemonoxacin against clinical isolates of Staphylococcus aureus: an in vitro comparison with three fluoroquinolones

In comparison with ciprofloxacin, levofloxacin and moxifloxacin, antimicrobial activity of nemonoxacin against ciprofloxacin-susceptible/-resistant methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) was determined with the availability to select resistant mutants evaluated. Minimum inhibitory concentrations and mutant prevention concentrations of quinolones were determined by agar dilution method, that concentrated bacterial cells were spread onto Mueller–Hinton agar plates containing antibacterials at different concentrations. Selection index (SI) was calculated. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.063 and 0.25 μg/mL for ciprofloxacin-susceptible MSSA and those were 0.5 and 4.0 μg/mL for ciprofloxacin-resistant MSSA, lower than observations of three fluoroquinolones distinctly. SI of nemonoxacin and moxifloxacin were similar, with narrower mutant selective window than levofloxacin and ciprofloxacin. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.25 and 2.0 μg/mL for ciprofloxacin-susceptible MRSA, which were 0.5 and 16.0 μg/mL for ciprofloxacin-resistant MRSA. Values were lower than those determined from fluoroquinolones. Nemonoxacin presents good antimicrobial activity against clinical isolates of S. aureus, especially for ciprofloxacin-resistant strains. But stepwise mutant accumulation of ciprofloxacin-resistant MRSA can be hardly inhibited by nemonoxacin with pharmacokinetic parameters considered.     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

The prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus in milk and dairy products in Riyadh,Saudi Arabia

In terms of life- menaced contagion, methicillin resistant Staphylococcus aureus (MRSA) is known to be one of which and it is truly notable in the contaminated food causing a community health anxiety. However, the occurrence of S. aureus and MRSA in diverse kinds of dairy products have been tested in this study. Samples from: raw milk (unpasteurized) from horse, goat, camel, and cow origins and unpacked cheese were checked for the recovered strains of such bacterium and MRSA. Wholly, MRSA isolates were verified for antimicrobial susceptibility and further characterized by mecA and staphylococcal cassette chromosome mec (SCCmec) typing. Also, Panton-Valentine Leukocidin (PVL), Staphylococcus aureus protein A (spa), and Staphylococcal enterotoxins (SEs) were also tested between all positive MRSA isolates in order to discover the virulence factors. Consequently, 70% of the 100 collected dairy products samples were contaminated by S. aureus bacteria and 72.9% of them were defined as MRSA. 9.8% of MRSA isolates contained mecA genes with SCCmec type II (80%) as the most common SCCmec type. Moreover, large number of MRSA isolates were identified as multidrug resistance and 28.6% of MRSA-mecA positive isolates were also carried vancomycin resistance genes (i.e., vanB). Too, spa gene was detected between 9.8% of MRSA isolates but PVL gene was not spotted at all. Additionally, the existing of SEs was variable between MRSA isolates and the most common type was SEH (51%). In general, our results confirmed that raw milk and unpacked cheese in the Kingdom of Saudi Arabia (Riyadh) is a potential vehicle for multidrug resistant MRSA transmission. It is a critical civic health menace and stresses, thus; the need of applying well cleanliness practices is essential.     

Silver Nanoparticles Toxicity and Bactericidal Effect Against Methicillin-Resistant Staphylococcus aureus: Nanoscale Does Matter

Silver nanoparticles, which are being used increasingly as antimicrobial agents, may extend its antibacterial application to methicillin-resistant Staphylococcus aureus (MRSA), the main cause of nosocomial infections worldwide. To explore the antibacterial properties of silver nanoparticles against MRSA, the present work includes an analysis of the relation between nanosilver effect and MRSA’s resistance mechanisms, a study of the size dependence of the bactericidal activity of nanosilver and a toxicity assessment of nanoparticles against epithelial human cells. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and MBC/MIC ratio of silver nanoparticles were quantified by using a luciferase-based assay. The cytotoxic effect (CC₅₀ and CC₉₀) of three different nanosilver sizes (10, 30–40, and 100 nm) were assessed in HeLa cells by a similar method. The therapeutic index was used as an indicator of nanosilver overall efficacy and safety. Silver nanoparticles inhibited bacterial growth of both MRSA and non-MR S. aureus in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio?≤?4). Silver nanoparticle’s therapeutic index varied when nanoparticle’s size diminished. At the same dose range, 10 nm nanoparticles were the most effective since they did not affect HeLa’s cell viability while inhibiting a considerable percentage of MRSA growth. Silver nanoparticles are effective bactericidal agents that are not affected by drug-resistant mechanisms of MRSA. Nanosilver size mediates MRSA inhibition and the cytotoxicity to human cells, being smaller nanoparticles the ones with a better antibacterial activity and nontoxic effect.     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11–0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4?×?MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6–45.6 and 1.7–3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI?=?0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI?=?0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI?=?0.125–0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI?=?8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.     

Design, expression, and characterization of a novel targeted plectasin against methicillin-resistant Staphylococcus aureus

A novel specifically targeted antimicrobial peptide (STAMP) that was especially effective against methicillin-resistant Staphylococcus aureus (MRSA) was designed by fusing the AgrD1 pheromone to the N-terminal end of plectasin. This STAMP was named Agplectasin, and its gene was synthesized and expressed in Pichia pastoris X-33 via pPICZαA. The highest amount of total secreted protein reached 1,285.5 mg/l at 108 h during the 120-h induction. The recombinant Agplectasin (rAgP) was purified by cation exchange chromatography and hydrophobic exchange chromatography; its yield reached 150 mg/l with 94 % purity. The rAgP exhibited strong bactericidal activity against S. aureus but not Staphylococcus epidermidis or other types of tested bacteria. A bactericidal kinetics assay showed that the rAgP killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in both Mueller–Hinton medium and human blood within 10 h when treated with 4× minimal inhibitory concentration. The rAgP caused only approximately 1 % hemolysis of human blood cells, even when the concentration reached 512 μg/ml, making it potentially feasible as a clinical injection agent. In addition, it maintained a high activity over a wide range of pH values (2.0–10.0) and demonstrated a high thermal stability at 100 °C for 1 h. These results suggested that this STAMP has the potential to eliminate MRSA strains without disrupting the normal flora.     

Fast DNA isolation and PCR protocols for detection of methicillin-resistant staphylococci

A rapid method that included simple boiling DNA extraction followed by a fast polymerase chain reaction (PCR) cycling protocol designed to detect mecA, which characterizes methicillin-resistant Staphylococcus aureus (MRSA), was performed. Briefly, the PCR cycling protocol consisted of pre-denaturation at 95 °C for 30 s, 30 cycles of denaturation at 94 °C for 2 s, annealing at 52 °C for 5 s, extension for 10 s, and final extension at 72 °C for 1 min. A good level of reliability of the method was verified. The study has shown that the method described here represents a rapid and accurate DNA extraction and PCR-based identification system of MRSA, thus allowing clinicians to make early identification and early implementation of control measures.     

Genotypic characterization of methicillin-resistant Staphylococcus aureus strains isolated from the anterior nares and catheter of ambulatory hemodialysis patients in Mexico

Methicillin-resistant Staphylococcus aureus (MRSA) is the causal agent of multiple nosocomial infections worldwide, including catheter-associated bacteremia in hemodialysis patients. The purposes of this work were to genetically characterize a group of MRSA isolates from catheter-related infections of ambulatory Mexican hemodialysis patients and to determine whether the strains are the same as those carried by the patients in their anterior nares. Sixteen pairs of MRSA isolates from the catheter (cat) and anterior nares (N) of hemodialysis patients were compared using pulsed-field gel electrophoresis (PFGE), PCR detection of adhesion genes and other virulence markers, and an antibiogram. Three pairs of N/cat MRSA isolates (18.7 %) with identical resistograms also showed the same combination of PCR-detected markers and PFGE pattern; one additional pair showed only an identical electrophoretic PFGE pattern. Of the MRSA isolates, 75 % (n?=?24) were resistant to ≥7 antibiotics, 4 isolates were resistant to 11 antibiotics, and 7 isolates were resistant to the 12 antibiotics tested. The most frequent virulence marker combination found was spa, clfA, clfB, cna, bbp, ebps, map/eap, sdrC, sdrD, sdrE, ica, agr (65.6 %, n?=?21). The SCCmec alleles of the 32 MRSA isolates were IV (n?=?20), I (n?=?7), II (n?=?4), and V (n?=?1), and no SCCmec type III MRSA was found. The genotypic characterization of the MRSA isolates studied in this work will contribute to a better understanding of the virulence gene makeup of catheter-colonizing S. aureus strains and will help to lower the infection risk in these patients.     

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30 °C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30 °C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40 °C and 50 °C respectively.     

Development and characterization of essential oil component-based polymer films: a potential approach to reduce bacterial biofilm

The development of new polymeric materials aimed to control the bacterial biofilm appears to be an important practical approach. The goal of the present study was to prepare and characterize poly(ethylene-co-vinyl acetate) copolymer (EVA) films containing citronellol, eugenol, and linalool and evaluate their efficiency on growth and biofilm formation of Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa in monospecies and dual species. The results showed that the addition of oil components influenced the elastic modulus (15 % decrease), the tensile stress (30 % decrease), the elongation at break (10 % increase), and the contact angle values (10–20° decrease) while leaving the homogeneity of the surface unaltered. Among the polymeric films, EVA + citronellol and EVA + eugenol at 7 wt% had the best inhibitory effect. After 24–48 h of incubation, EVA + citronellol was more effective against the growth (30–60 % reduction) than EVA + eugenol (15–30 % inhibition). However, this inhibition decreased after 240 h of incubation. On the contrary, the biofilm evaluation revealed a strong inhibition trend also after prolonged incubation time: the amount of biomass per square centimeter formed on copolymer with oil components was significantly less (40–70 % decrease) than that on pure copolymer control for L. monocytogenes, S. aureus, and E. coli. When polymeric materials were simultaneously inoculated with combinations of S. aureus and E. coli, the biomass accumulated was higher for EVA + citronellol and lower for EVA + eugenol than that in monoculture biofilm. The findings were similar to the results obtained by 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay that measures the metabolic activity of viable cells.     

Uric Acid Excretion in Isoparorchis hypselobagri (Billet, 1898)

The nature and amount of excretory products of Isoparorchis hypselobagri, a digenetic trematode inhabiting the swim bladder of Wallago attu have been studied. The parasites were kept in PBS media without glucose (control) and with glucose (treated) successfully up to 300 h and different excretory products were estimated at an interval of 12 h. The quantitative estimation of excreted uric acid in I. hypselobagri was revealed less in amount in the flukes which were kept in vitro in the control incubation media than those considered from the treated incubation media. The highest amount of uric acid excreted in control media was 6.72 mg % at 204 h, and in treated condition 16.658 mg % after 216 h of incubation. The lowest amount of uric acid excreted, 0.186 mg % in control media after 252 h of incubation and 2.896 mg % in treated media after 192 h of incubation. The rate of excretion of uric acid in treated condition after every 12 h of incubation is much higher than the control. Significant amount of uric acid have also been recorded in the swim bladder washings of host. It is apparent that the amount of excreted uric acid depends on the number and weight of the parasites harbor. Results suggest that I. hypselobagri is ammonotelic, ureotelic and also uricotelic trematode.     

Optical forward-scattering for identification of bacteria within microcolonies

The development of methods for the rapid identification of pathogenic bacteria is a major step towards accelerated clinical diagnosis of infectious diseases and efficient food and water safety control. Methods for identification of bacterial colonies on gelified nutrient broth have the potential to bring an attractive solution, combining simple optical instrumentation, no need for sample preparation or labelling, in a non-destructive process. Here, we studied the possibility of discriminating different bacterial species at a very early stage of growth (6 h of incubation at 37 °C), on thin layers of agar media (1 mm of Tryptic Soy Agar), using light forward-scattering and learning algorithms (Bayes Network, Continuous Naive Bayes, Sequential Minimal Optimisation). A first database of more than 1,000 scatterograms acquired on 7 gram-negative strains yielded a recognition rate of nearly 80 %, after only 6 h of incubation. We investigated also the prospect of identifying different strains from a same species through forward scattering. We discriminated, thus, four strains of Escherichia coli with a recognition rate reaching 82 %. Finally, we show the discrimination of two species of coagulase-negative Staphylococci (S. haemolyticus and S. cohnii), on a commercial selective pre-poured medium used in clinical diagnosis (ChromID MRSA, bioMérieux), without opening lids during the scatterogram acquisition. This shows the potential of this method—non-invasive, preventing cross-contaminations and requiring minimal dish handling—to provide early clinically-relevant information in the context of fully automated microbiology labs.     

Construction of an RNAi expression vector and transformation into Penicillium chrysogenum

In this work, we constructed an RNAi vector for attenuation of the class III chitin synthase gene chs4, which plays a major role in hyphal growth and conidia formation. To achieve a high transformation frequency, factors affecting the preparation and regeneration of protoplasts were analyzed. The maximum numbers of protoplasts (1.41?×?10⁷ mL^?1) were released when mycelia cultured for 48 h were incubated at 30 °C for 5 h in a buffer containing 4 mg mL^?1 lysing enzyme. The maximum regeneration rate (33 %) was obtained when mycelia were digested for 4 h and plated on a regeneration medium containing 1 % overlaid agar. Quantitative real-time PCR was performed to validate the transformation efficiency, and it revealed knockdown of chs4 gene in randomly selected transformants at different levels. Dramatic reductions in the formation of conidia and the hyphal growth rate were observed in most of the transformants.     

Improved microspore embryogenesis induction and plantlet regeneration using putrescine,cefotaxime and vancomycin in Brassica napus L.

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l^?1) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l^?1 putrescine. Putrescine treatment at 0.5 mg l^?1 for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish^?1) was possible when induction medium was supplemented with 50 mg l^?1 cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l^?1 at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l^?1 cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l^?1 during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish^?1) in contrast to untreated cultures (93.6 embryos Petri dish^?1) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l^?1 for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.     

Urea Excretion in Isoparorchis hypselobagri (Billet, 1898)

To understand the nature and amount of excretory products of Isoparorchis hypselobagri, a digenetic trematode inhabiting the swim bladder of Wallago attu the parasites were kept in PBS media without glucose (control) and with glucose (treated) successfully up to 300 h and estimated different excretory products in the media after every alternate 12 h. The quantitative estimation of urea excreted by I. hypselobagri is done. It is lower when the flukes were kept in vitro in the control incubation media than in the treated incubation media. The highest amount of urea excreted in control media was 18.563 mg % at 276 h, and in treated condition 29.759 mg % after 216 h of incubation, while the lowest amount of urea excreted, 6.08 mg %, in control media and 10.343 mg % in treated media respectively after 12 h of incubation. The results were highly significant at 5 % level. The rate of change of excretion of urea in treated condition after every 12 h of incubation time interval was also studied. In the swim bladder washings of host urea is also present in significant amount and the amount excreted depends on the number of parasites harbor. From the results presence of both ammonotelic and ureotelic conditions are suggested.     

Cloning, Expression, and Characterization of Iron Superoxide Dismutase in Sonneratia alba, a Highly Salt Tolerant Mangrove Tree

In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5–9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.