Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Metabolism of Atrazine in Liquid Cultures and Soil Microcosms by Nocardioides Strains Isolated from a Contaminated Nigerian Agricultural Soil

Atrazine-degrading microorganisms designated EAA-3 and EAA-4, belonging to the genus Nocardioides, were obtained from an agricultural soil in Nigeria. The degradation kinetics of the two strains revealed total disappearance of 25 mg l^?1 of atrazine in less than 72 h of incubation at the rate of 0.42 mg l^?1 h^?1 and 0.35 mg l^?1 h^?1, respectively. Screening for atrazine catabolic genes in these organisms revealed the presence of trzN, atzB, and atzC. Other genes, specifically atzA, atzD, and trzD, were not detected. Potential intermediates of atrazine catabolic route such as hydroxyatrazine, desethylatrazine, and desisopropylatrazine were utilized as sources of carbon and energy, while desisopropyl desethyl-2-hydroxyatrazine and desisopropyl-2-hydroxyatrazine were attacked but in the presence of glucose. A soil microcosm study showed that degradation was faster in microcosms contaminated with 13 mg of atrazine per g^?1 of soil compared with 480 mg g^?1 of soil. In the former, degradation was 10% higher in the inoculated soil than the non-inoculated control (natural attenuation) over the 28-day study period. Corresponding value obtained for the latter was nearly 70% higher. This study has demonstrated that the bacterial strains isolated enhanced atrazine degradation and the catabolic activities of these strains were not affected with increasing soil atrazine concentration.     

Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants

Abstract

Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for their anticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenols and ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petiole and root explants are reported. Callus induction and proliferation depended on the genotype and the experimental conditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on medium supplemented with 2,4-D (0.1 mg l^?1) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l^?1) plus 6-benzil-ammino-purine (BAP) (0.22 mg l^?1) for A. sedifolius. The content of total polyphenol and ascorbic acid was estimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lower than in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reduced ascorbic acid pool increased. These findings demonstrate that Aster callus cultures produce antioxidant compounds and as such might be a model system to investigate the regulation and production of these important metabolites.     

In vitro plant regeneration from leaf callus of Grindelia robusta Nutt

Abstract

A regeneration protocol from leaf explants of Grindelia robusta Nutt. was developed. The combination of 0.5 mg l^?1 IBA plus 0.5 mg l^?1 or 1 mg l^?1 BA added to Murashige-Skoog (MS) medium resulted in the best callus induction frequency; the combination of 0.4 or 0.9 mg l^?1 BA plus 1.2 mg l^?1 GA₃ resulted in the best shoot regeneration. Rooting was successful on MS medium supplemented with 0.5 mg l^?1 IBA. Hardening of G. robusta plants was accomplished in 30 days with 85% survival rate.     

Rapid treatment of vessels fouled with an invasive polychaete,Sabella spallanzanii,using a floating dock and chlorine as a biocide

Chlorine solution was added to the water encapsulated within a proprietary ‘floating dock’ to treat a vessel infested with the invasive polychaete Sabella spallanzanii. The chlorine was added as sodium dichloroisocyanurate (‘dichlor’) at an initial concentration of 200 mg l^?1 of free available chlorine (FAC). This concentration killed 99% of S. spallanzanii in their tubes during a 4-h exposure in laboratory tests (EC₉₉ 160 mg FAC l^?1). The concentration of FAC in the floating dock declined to ~50 mg l^?1 after 4 h and < 10 mg l^–1 after 16 h. Residual FAC was neutralised with thiosulphate at completion of exposure. A sample of 30 S. spallanzanii individuals collected from the hull after treatment all showed morphological damage and 28 showed no response to touch. Re-examination of the hull after 6 d found no live worms or other fouling organisms. This method provides a cost-effective, rapid means of treating hull fouling.     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l^?1 benzyladenine (BA) and 0.1 mg l^?1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l^?1 BA along with 1.0 mg l^?1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l^?1 thidiazurone (TDZ) and 0.1 mg l^?1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l^?1 BA and 0.1 mg l^?1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.     

Biofouling control using microparticles carrying a biocide

This study presents a new technological approach to minimize the use of antimicrobial (AMB) agents and their deleterious effects, based on the principle of drug-delivery systems whereby the AMB chemicals are transported on microparticles. The efficacy of microparticles carrying the quaternary ammonium compound (QAC), benzyldimethyldodecyl ammonium chloride (BDMDAC), was assessed against Pseudomonas fluorescens in both the planktonic and the biofilm state. The microparticles were prepared using a layer-by-layer (LBL) self-assembly technique. Oppositely charged molecules of polyethyleneimine (PEI), sodium polystyrene sulfonate (PSS), and BDMDAC were assembled on polystyrene (PS) cores. BDMDAC-coated particles were observed by CryoSEM and their composition analyzed by X-ray microanalysis. Zeta potential measurements indicated that changes in surface charge were compatible with a BDMDAC/particle interaction. This biocidal carrier structure had significant stability, verified by the release of only 15% of the BDMDAC when immersed in water for 18 months. Biocidal carrier activity was evaluated by determining the survival ratio of P. fluorescens planktonic and biofilm cells after different exposure periods to BDMDAC-coated particles. Tests with biofilm cells were also performed with the free QAC. An efficient AMB effect (minimum bactericidal concentration) against suspended cells was found for a concentration of 9.2 mg l^?1 of BDMDAC on coated particles after incubation for 30 min and 6.5 mg l^?1 of BDMDAC on coated particles after 60 min. Exposure of biofilms to PS-PEI/PSS/BDMDAC (0.87 mg l^?1) resulted in a decrease in viability of 60.5% and 66.5% of the total biofilm population for 30 and 60 min exposure times, respectively. Exposure for 60 min to 6.33 mg l^?1 and 11.75 mg l^?1 of BDMDAC in PS-PEI/PSS/BDMDAC particles promoted inactivation of 80.6% and 87.2% of the total population, respectively. The AMB effects obtained with the application of free BDMDAC were statistically similar to those promoted by the application of BDMDAC coated particles. The overall results indicate that this novel AMB strategy has potential for the control of microbial growth of planktonic cells and biofouling. Moreover, the technique allows the reuse of AMB molecules and consequently reduces the environmental risks associated with excessive use of AMB agents, thereby providing real benefits to public health.     

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l^?1, while the minimum biofilm elimination concentration (MBEC) was 780–3,120 mg l^?1. Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg?l^?1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg?l^?1 TDZ. The WPM medium supplemented with 2.5 mg?l^?1 BA?+?0.4 mg?l^?1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg?l^?1 BA and 3.0 mg?1^?1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg?l^?1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.     

Copper oxide nanoparticle toxicity in mung bean (Vigna radiata L.) seedlings: physiological and molecular level responses of in vitro grown plants

In this study, the toxic effect of copper oxide nanoparticles (CuONPs) at the physiological and molecular level was investigated in mung bean (Vigna radiata L.) plants. The seedlings were grown in half strength Murashige and Skoog medium supplemented with different concentrations of CuONPs (0, 20, 50, 100, 200 and 500 mg l^?1) for 21 days under controlled growth conditions. Exposure to 200 and 500 mg l^?1 of CuONPs significantly reduced shoot length and biomass. Significant reduction in root length and biomass was observed upon exposure to all concentrations of CuONPs. Retardation of primary and lateral root growth was observed upon exposure to different concentrations of CuONPs. At 100, 200 and 500 mg l^?1 of CuONPs exposure, the total chlorophyll contents reduced significantly. Exposure to different concentrations of CuONPs has not resulted in any significant change in carotenoid contents. The proline content significantly increased upon exposure to 100, 200 and 500 mg l^?1 of CuONPs. Significant increase in hydrogen peroxide content and lipid peroxidation was observed in roots upon exposure to 20, 50, 100, 200 and 500 mg l^?1 of CuONPs. Histochemical staining with nitroblue tetrazolium and treatment with 3′-(p-hydroxyphenyl) fluorescein indicated a concentration-dependent increase in reactive oxygen species generation in roots. Exposure to CuONPs has resulted in excess lignification of roots cells as revealed by phloroglucionol-HCl staining. Gene expression analysis using real-time polymerase chain reaction showed modulations in the expression of CuZn superoxide dismutase, catalase and ascorbate peroxidase genes in roots of CuONPs exposed plants.     

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.     

Individual- and population-level effects of antibiotics on the rotifers, <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> and <Emphasis Type="Italic">B. plicatilis</Emphasis>

The toxicity of three common antibiotics (streptomycin sulfate, tetracycline hydrochloride, and tylosin tartrate) to the freshwater rotifer Brachionus calyciflorus and brackish-water rotifer B. plicatilis was investigated using full-lifespan exposure durations. Effects of each antibiotic on lifespan, lifetime reproduction, and Malthusian parameter were assessed at seven nominal concentrations (ranging from 5.6 mg l⁻¹ to 2,000 mg l⁻¹) and a negative control. Lowest Observed Effect Concentrations (LOECs) were determined for reproduction and lifespan, while 1%, 10%, 25%, and 50% Inhibitory Concentrations (IC₁, IC₁₀, IC₂₅, IC₅₀) and 95% confidence intervals were estimated for all three endpoints. LOECs ranged from 5.6 mg l⁻¹ to 90 mg l⁻¹, with all LOECs less than 90 mg l⁻¹ occurring in B. calyciflorus. The lowest IC1 concentrations were 3.91 mg l⁻¹ for the effect of tetracycline on lifetime reproduction in B. calyciflorus and 4.06 mg l⁻¹ for the effect of tylosin on lifetime reproduction in B. plicatilis. Overall, lifetime reproduction was the most sensitive endpoint and the Malthusian parameter was the least sensitive. IC₁ values for lifetime reproduction were roughly one to two orders of magnitude lower than the corresponding IC₅₀ values. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Morphological,anatomical, and ultrastructural changes (visualized through scanning electron microscopy) inducedin Triticum aestivum by Pb2+ treatment

Lead (Pb) causes severe damage to crops, ecosystems, and humans, and alters the physiology and biochemistry of various plant species. It is hypothesized that Pb-induced metabolic alterations could manifest as structural variations in the roots of plants. In light of this, the morphological, anatomical, and ultrastructural variations (through scanning electron microscopy, SEM) were studied in 4-day-old seedlings of Triticum aestivum grown under Pb stress (0, 8, 16, 40, and 80 mg Pb²⁺ l^?1; mild to highly toxic). The toxic effect was more pronounced in radicle growth than on the plumule growth. The SEM of the root of T. aestivum depicted morphological alterations and surface ultrastructural changes. Compared to intact and uniform surface cells in the control roots, cells were irregular and desiccated in Pb²⁺-treated roots. In Pb²⁺-treated roots, the number of root hairs increased manifold, showing dense growth, and these were apparently longer. Apart from the deformity in surface morphology and anatomy of the roots in response to Pb²⁺ toxicity, considerable anatomical alterations were also observed. Pb²⁺-treated root exhibited signs of injury in the form of cell distortion, particularly in the cortical cells. The endodermis and pericycle region showed loss of uniformity post Pb²⁺ exposure (at 80 mg l^?1 Pb²⁺). The cells appeared to be squeezed with greater depositions observed all over the tissue. The study concludes that Pb²⁺ treatment caused structural anomalies and induced anatomical and surface ultrastructural changes in T. aestivum.     

Augmentin<Superscript>®</Superscript> as an alternative antibiotic for growth suppression of <Emphasis Type="Italic">Agrobacterium</Emphasis> for tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) transformation

Augmentin^® was used as an alternative antibiotic for suppressing Agrobacterium tumefaciens during tomato (Lycopersicon esculentum) transformation. The efficiency of Augmentin compared with Timentin^® to suppress the growth of Agrobacterium was at concentrations of 300 and 100 mg l^–1, respectively. In addition, the high concentration up to 500 mg l^–1 of both antibiotics showed no significant toxicity to shoot regeneration. The overgrowth of Agrobacterium was observed on tomato shoots regenerated on medium containing cefotaxime even at high concentration up to 500 mg l^–1. Moreover, shoot regeneration from medium containing cefotaxime was lower than the one from medium containing Timentin and Augmentin. However, we found that Timentin was more stable than Augmentin when stored as a mixed stock solution and kept at –20°C freezer. In order to increase the efficiency of Augmentin, we recommended the use of Augmentin at a concentration of 300 mg l^–1 with fresh preparation prior to use. Therefore, Augmentin can be used as another antibiotic for suppression of bacterial growth with comparative efficiency as Timetin for tomato transformation.     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l^?1), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD® BacLight?). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l^?1 of free chlorine and in 10 min when the concentration is increased to 1.2 mg l^?1. However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.     

Lindane degradation by root epiphytic bacterium Achromobacter sp. strain A3 from Acorus calamus and characterization of associated proteins

Abstract

Lindane degrading root epiphytic bacteria were isolated from wetland plant Acorus calamus. Bacterial strain A3 identified as Achromobacter sp. A3, showed maximum degradation potential of 88.7?±?1.24% for 50?mg?l^?1 lindane. Lindane biodegradation was followed by decrease in pH as well as increase in concentration of chloride ions in the culture medium. Lindane degradation potential of Achromobacter sp. A3 was also studied at different concentrations of lindane. Maximum degradation was at 10?mg l^?1 followed by 50?mg l^?1 and 100?mg l^?1 lindane. Also, lindane induced proteins were studied using SDS-PAGE. The induced proteins were identified as alpha/beta hydrolase fold-3 domain-containing protein, involved in lindane hydrolysis and extracellular solute-binding family protein having role in transmembrane transport of lindane for utilization of lindane by bacteria. The appearance of unique polypeptides in lane corresponding to media supplemented with lindane showed that the exposure of bacterial cells to lindane has resulted in regulative expression of certain proteins. So far as known, this is the first report to isolate and study lindane degrading root epiphytic bacteria from A. calamus.