Opportunistic endophytism of Trichoderma species in rice Pusa Basmati-1 (PB1)

Microbes that colonize internal tissues of plants are called endophytes, andare known for their functional role against biotic/abiotic stress and growth promotion activity in plants. The ascomyceteous fungus Trichoderma spp. (Teleomorph: Hypocrea) are well known antagonists cum biocontrol agents. In this study, a total of five Trichoderma isolates from two different species viz., T. asperellum (TaR1, TaR2 and TaR3) and T. asperelloides (TaR4 and TaR5) collected from different agro-climatic zones in Rajasthan, India were evaluated for their endophytism in rice variety Pusa Basmati-1 (PB-1) through soil and seed treatment. An attempt was made to re-isolate the fungus from rice roots (seed and soil treated) and further subjected to microscopic and molecular analysis. Re-isolation results revealed that culture growth of Trichoderma spp. isolated was similar to that of the inoculated ones. The microscopic analysis (light and scanning electron microscopy) results also confirmed that the re-isolated endophytic fungus were identical to the inoculated ones. These results were further confirmed by polymerase chain reaction (PCR) amplification of the rDNA region (18SrRNA, ITS1, 5.8SrRNA, ITS2 and 28SrRNA) and translation elongation factor 1 (Tef1) with the re-isolated Trichoderma asperellum and T. asperelloides isolates. In this study, it has been confirmed that Trichoderma asperellum and T. asperelloides turns endophytic in rice after introduction through seed and soil treatment.

Molecular response to imatinib & its correlation with mRNA expression levels of imatinib influx & efflux transporters in patients with chronic myeloid leukaemia in chronic phase.

Background & objectives: Imatinib is the standard first-line treatment for chronic myeloid leukaemia (CML) patients. About 20 to 30 per cent patients develop resistance to imatinib and fail imatinib treatment. One of the mechanisms proposed is varying expression levels of the drug transporters. This study was aimed to determine the expression levels of imatinib transporter genes (OCT1, ABCB1, ABCG2) in CML patients and to correlate these levels with molecular response. Methods: Sixty three CML chronic phase patients who were on 400 mg/day imatinib for more than two years were considered for gene expression analysis study for OCT1, ABCB1 and ABCG2 genes. These were divided into responders and non-responders. The relative transcript expression levels of the three genes were compared between these two categories. The association between the expression values of these three genes was also determined. Results: No significant difference in the expression levels of OCT1, ABCB1 and ABCG2 was found between the two categories. The median transcript expression levels of OCT1, ABCB1 and ABCG2 genes in responders were 26.54, 10.78 and 0.64 versus 33.48, 7.09 and 0.53 in non-responders, respectively. A positive association was observed between the expression of the ABCB1 and ABCG2 transporter genes (r=0.407, P<0.05) while no association was observed between the expression of either of the ABC transporter genes with the OCT1 gene. Interpretation & conclusions: Our findings demonstrated that the mRNA expression levels of imatinib transporter genes were not correlated with molecular response in CML patients. further studies need to be done on a large sample of CML patients to confirm these findings.

Isolation of genomic DNA from medicinal plants without liquid nitrogen.

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the λ260/λ280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.

Antimicrobial resistance in Enterococcus faecalis at a tertiary care centre of northern India.

BACKGROUND & OBJECTIVES: Multiresistant enterococci are emerging as a leading nosocomial pathogen. Knowledge of the profile of antimicrobial resistance is essential to formulate treatment guidelines for infections caused by enterococci. This study reports the antimicrobial sensitivity of enterococci isolated during a one year period from clinical samples of patients admitted to a teriary care hospital of Delhi. METHODS: A total of 444 isolates of Enterococcus faecalis were screened for antimicrobial susceptibility by the disk diffusion technique as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Screening for vancomycin resistance was done by the vancomycin screen agar method recommended by NCCLS, which was confirmed by determination of minimum inhibitory concentration (MIC) using microbroth dilution and E-test methods. Vancomycin resistance phenotypes were determined by polymerase chain reaction. RESULTS: A total of 115 (26%) isolates had high level aminoglycoside resistance, 293 (66%) were resistant to ampicillin, 391 (88%) to ciprofloxacin and 377 (85%) to erythromycin. Vancomycin resistance was found in five (1%) isolates, of which four had van A phenotype and one had van B phenotype. INTERPRETATION & CONCLUSION: Emergence of vancomycin resistant enterococci is of concern due to the limited therapeutic options. Implementation of infection control measures can contain the spread of these resistant bacteria.

Identification of Acinetobacter species isolated from clinical specimens by amplified ribosomal DNA restriction analysis.

BACKGROUND & OBJECTIVES: Taxonomy of Acinetobacter has been changing ever since it was recognized to be associated with human infections. Many biochemical schemes and molecular methods have been used for the species identification of this bacterium. Recently a simple molecular method called amplified ribosomal DNA restriction analysis (ARDRA) has been used to determine the genomospecies of ACINETOBACTER: An attempt is made in the present study to identify the Acinetobacter genomospecies isolated from clinical specimens using ARDRA and to see whether the environmental isolates are similar to those obtained from clinical specimens. METHODS: A total of 142 consecutive isolates of Acinetobacter sp. obtained from different clinical specimens (125) and environmental samples (17) of postoperative neurosurgery-intensive care unit were studied using ARDRA. Amplification was done using primers of 16S rRNA gene followed by restriction with Alu I, Cfo I and Mbo I enzymes separately to obtain a profile of patterns specific for a species. RESULTS: Of the 125 clinical isolates, 107 were Acinetobacter baumannii (genomospecies 2) and 18 were A. calcoaceticus (genomospecies 1); while 11 of the 17 environmental isolates were A. baumannii and 6 had unidentifiable patterns which were not found in the clinical isolates. INTERPRETATION & CONCLUSION: We found that ARDRA was a simple and reproducible method to be used in a clinical laboratory for identification of Acinetobacter species. A. baumannii was found to be the commonest species isolated from the patients and environment in our hospital. The presence of the same species of Acinetobacter in the environment suggests the role of environment as a source of infection to the patients in high risk units.