Efflux-pump system and biofilm formation are two important mechanisms Pseudomonas aeruginosa deploys to escape the effects of antibiotics. The current study was undertaken from September 2019 to March 2020 at a tertiary-care hospital in Kathmandu in order to ascertain the burden of P. aeruginosa in clinical specimens, examine their biofilm-forming ability and determine their antibiotic susceptibility pattern along with the possession of two efflux-pump genes-mexA and mexB. Altogether 2820 clinical specimens were collected aseptically from the patients attending the hospital and processed according to standard microbiological procedures. Identification of P. aeruginosa was done by Gram stain microscopy and an array of biochemical tests. All the P. aeruginosa isolates were subjected to in vitro antibiotic susceptibility testing and their biofilm-forming ability was also examined. Presence of mexA and mexB efflux-pump genes was analyzed by Polymerase Chain Reaction (PCR) using specific primers. Out of 603 culture positive isolates, 31 (5.14%) were found to be P. aeruginosa, of which 55% were multi-drug resistant (MDR). Out of 13 commonly used antibiotics tested by Kirby-Bauer disc diffusion method, greatest resistance was shown against piperacillin-tazobactam 15 (48.4%) and ceftazidime 15 (48.4%), and least against meropenem 6 (19.4%) and ofloxacin 5 (16.2%). Of all 17 MDR isolates subjected to biofilm detection, strong biofilm formation was exhibited by 11 (65%) and 14 (82%) isolates with microtiter plate method and tube method respectively. Out of 17 isolates tested, 12 (70.6%) isolates possessed mexA and mexB genes indicating the presence of active efflux-pump system. Higher number of the isolates recovered from sputum 7 (58.3%) and pus 5 (41.7%) possessed mexA/mexB genes while the genes were not detected at all in the isolates recovered from the urine (p<0.05). This study assessed no significant association between biofilm production and multi-drug resistance (p>0.05). Adoption of stern measures by the concerned authorities to curb the incidence of multi-drug resistant and biofilm-forming isolates is recommended to prevent their dissemination in the hospital settings.

Crown and root rot disease was among the major problems in acid lime (Citrus aurantifolia) seedlings grown in the nursery of Warm Temperate Horticulture Centre, Kirtipur, Nepal. Samples of the diseased plants were investigated at the laboratory of Nepal Plant Disease and Agro Associates. Rhizoctonia solani, Fusarium solani and Alternaria citri were found associated with the disease samples. On pathogenicity testing R. solani showed symptoms like those of the infected seedlings. Hence, a pot experiment was conducted in a completely randomized design with four replications to manage the disease. Acid lime seedlings grown in sterilized soil and inoculum of R. solani prepared in potato dextrose agar (PDA) were used for the experiment. The treatments included carbendazim 50WP @ 1000 ppm, Trichoderma viride @ 2 ml/l water, Pseudomonas fluorescens @ 2 ml/l water, A-arya 009 (essential oil and organic acids) @ 1 ml/l water, and control (water) which were applied by soil drenching before 15 days of pathogen inoculation. Carbendazim at 100 and 200 ppm was also evaluated against R. solani by poison food technique on PDA. In the pot culture, the effect of treatments was significant on the growth of root and shoot of seedlings and both disease incidence and severity. Carbendazim had significantly least disease incidence (43.75%) and least disease severity (18.75%) among the treatments 45 days after inoculation with the pathogen. Also, the fungicide at 100 ppm gave complete inhibition of R. solani in PDA culture. Hence, carbendazim could be suggested for the management of the citrus crown and root rot caused by R. solani. However, further study is required to find out appropriate concentration of the fungicide for effective management of disease.