Lower Susceptibility to Fluoroquinolones and a gyrA Gene Mutation in Salmonella Typhi Isolates from Enteric Fever Patients.

BACKGROUND: Enteric fever remains a major cause of morbidity and mortality in Nepal. The emergence of multi-drug resistant Salmonella is a challenge to the clinician to care for patients with enteric fever. This study assessed the antibiotic susceptibility of Salmonella Typhi isolated from enteric fever and the presence of gyrA? ?gene mutation at ser83 of S. Typhi. METHODS: Blood samples (n = 834) from suspected enteric fever patients were collected and cultured to identify Salmonella Typhi. Antimicrobial sensitivity test was performed by the modified Kirby Bauer disc diffusion method. The minimum ?inhibitory concentration (MIC) tests for ofloxacin and ciprofloxacin were examined by the agar dilution method. The gyrA gene was amplified by PCR and restriction enzyme digestion was performed to evaluate the ser83 mutation. RESULTS: Among 824 blood samples analyzed, 5.1% (42/824) were culture positive for S. Typhi. First-line antibiotics chloramphenicol and co-trimoxazole showed higher in-vitro efficacy compared to amoxicillin. Macrolides (azithromycin) and third-generation cephalosporins (ceftriaxone, cefixime, and cefotaxime) were highly effective against S. Typhi. Nalidixic acid resistance (NAR) was observed in 95.2% (40/42) isolates, among them, all (40/40) isolates harbored mutant gyrA gene at ser83. However, none of the nalidixic acid-sensitive Salmonella? isolates was positive for gyrA? mutation at ser83. CONCLUSIONS: This study showed decreased susceptibility to fluoroquinolones and the presence of gyrA? mutation at ser83 position in majority of S. Typhi isolates which highlights the importance of alternate drugs as empirical therapy for the treatment of enteric fever patients. So, the clinician should focus on prescribing conventional first-line antibiotics for the treatment of typhoid patients after higher cohort and extended follow-up studies.

Detection of Bacillus Species with Arsenic Resistance and Plant Growth Promoting Efficacy from Agricultural Soils of Nepal.

Arsenic contamination in soil and water is one of the major environmental problems in multiple countries including Nepal imposing a serious threat to the ecosystem and public health. Many soil bacteria can detoxify arsenic, including genus Bacillus. With an objective to gauge the plant growth-promoting activities of arsenic-resistant Bacillus species, 36 samples (soil, rice, cauliflower, and beans) were collected from the Terai region of Nepal. For selective isolation of Bacillus species, each sample was heated at 80°C for 15 min before the inoculation into nutrient agar (NA). Following the standard protocol, arsenic-resistant Bacillus species were screened using NA supplemented with 100 ppm sodium arsenate and sodium arsenite. Among 158 randomly selected isolates, only five isolates were able to tolerate sodium arsenite concentration up to 600 ppm. Notably, all five isolates were able to produce indole acetic acid (IAA), a plant hormone, and solubilize phosphate. Based on biochemical analysis and 16S rRNA gene sequencing, isolates N4-1, RW, KR7-12, Bhw1-4, and BW2-2 were identified as B. subtilis subsp. stercosis, B. flexus, B. licheniformis, B. cereus, and B. flexus, respectively. To the best of our knowledge, this is the first study showing the presence of arsenic-resistant B. flexus in Nepalese soil with plant growth-promoting traits. Possible utilization of these Bacillus strains could facilitate the novel bioremediation pathway to reduce the toxic effect of arsenic from the soil and water in the Terai region of Nepal.

Clinical Specimens are the Pool of Multidrug- resistant Pseudomonas aeruginosa Harbouring oprL and toxA Virulence Genes: Findings from a Tertiary Hospital of Nepal.

The multidrug- or extensively drug-resistant (MDR/XDR) Pseudomonas aeruginosa carrying some virulence genes has become a global public health threat. However, in Nepal, there is no existing report showing the prevalence of oprL and toxA virulence genes among the clinical isolates of P. aeruginosa. Therefore, this study was conducted for the first time in the country to detect the virulence genes (oprL and toxA) and antibiotic susceptibility pattern of P. aeruginosa. A total of 7,898 clinical specimens were investigated following the standard microbiological procedures. The antibiotic susceptibility testing was examined by the modified disc diffusion method, and virulence genes oprL and toxA of P. aeruginosa were assessed using multiplex PCR. Among the analyzed specimens, 87 isolates were identified to be P. aeruginosa of which 38 (43.68%) isolates were reported as MDR. A higher ratio of P. aeruginosa was detected from urine samples 40 (45.98%), outpatients' specimens 63 (72.4%), and in patients of the age group of 60-79 years 36 (41.37%). P. aeruginosa was more prevalent in males 56 (64.36%) than in female patients 31 (35.63%). Polymyxin (83.90%) was the most effective antibiotic. P. aeruginosa (100%) isolates harboured the oprL gene, while 95.4% of isolates were positive for the toxA gene. Identification of virulence genes such as oprL and toxA carrying isolates along with the multidrug resistance warrants the need for strategic interventions to prevent the emergence and spread of antimicrobial resistance (AMR). The findings could assist in increasing awareness about antibiotic resistance and suggest the judicious prescription of antibiotics to treat the patients in clinical settings of Nepal.

Screening and characterization of potent poly glutamic acid producing Bacillus sp. isolated from Kinema, water and soil samples.

Microbially produced gamma poly glutamic acid (γ-PGA) is a commercially important biopolymer with many applications in foods and various other substances and are abundantly used in different parts of the world. With an aim to study the potent γ-PGA producing Bacillus species, a total of 47 different samples (Kinema, soil, and water) were randomly collected from different locations across the country, and Bacillus sp. were selectively isolated, screened, and characterized by performing physiological, biochemical, morphological, and 16S rRNA gene sequencing. The microbial production of γ-PGA was assayed with the selected isolates on the PGA medium and the metabolite obtained was recovered by ethanol precipitation method and further characterized by thin-layer chromatography (TLC). Thermotolerance (25-60 °C), pH tolerance (4-9), and NaCl tolerance (1-9%) tests were performed to optimize the bacterial growth and γ-PGA production and its viscosity were measured by Ostwald's viscometer. Out of 145 randomly selected colonies, 63 isolates were Gram-positive, rods, and endospore producers and were presumptively confirmed as genus Bacillus. Higher growth of γ-PGA producers were reported in 22 isolates and was found at optimum conditions such as temperature (30-37 °C), pH (6.5-7), incubation time (3 days), and NaCl concentration (3%) and γ-PGA thus produced was further verified by TLC with the retention factor (RF) value 0.27. The potent isolates were closely similar to Bacillus subtilis subsp. stercoris, Bacillus cereus, Bacillus paranthracis, and Bacillus licheniformis etc. Based on the findings of the study, B. licheniformis is the most potent γ-PGA producing Bacillus sp. which can further be used for the commercial production of γ-PGA. To the best of our knowledge, there is yet no published research from Nepal showing the production of the γ-PGA although microbially produced γ-PGA are the major constituents in some popular foods in particular communities of the country.

Prevalence of CTX-M ß-Lactamases Producing Multidrug Resistant Escherichia coli and Klebsiella pneumoniae among Patients Attending Bir Hospital, Nepal.

The emergence of multidrug resistant (MDR) bacteria which is attributable to extended spectrum ß-lactamases (ESBLs) production of CTX-M types is an obvious problem worldwide. This study is aimed at determining the prevalence of CTX-M ß-lactamases producing multidrug resistant Escherichia coli and Klebsiella pneumoniae among patients attending Bir Hospital. A cross-sectional study was conducted between April and September 2019 at Bir Hospital, Kathmandu, and Department of Microbiology, National College, Kathmandu, Nepal. A total of 5,690 different clinical specimens were subjected to cultural, microscopic, and biochemical analyses for the identification of the isolates. Antimicrobial susceptibility testing of the isolates was done, and MDR isolates were selected and processed for further ESBL confirmation by the combination disks method. All confirmed ESBL isolates were screened for CTX-M type ß-lactamases (bla CTX-M) by PCR. Of the total 345 isolates (227 Escherichia coli and 118 Klebsiella pneumoniae), 232 were MDR. All 232 (67.24%) MDR isolates were suspected as ESBL producers on the screening test. However, on the phenotypic test, 135 (58.18%) of total MDR bacteria were confirmed as ESBL producers with the highest proportion in K. pneumoniae (59.37%). The major source of ESBL producers was urine. ESBL producing isolates were mostly identified from outpatients and patients belonging to age group 41-60. Gentamicin was found to be effective against ESBL producers. The prevalence of bla CTX-M was (89.62%) with the highest frequency for E. coli (93.81%). High prevalence of ESBL of CTX-M types among MDR E. coli and K. pneumoniae was detected from clinical specimens of patients in Bir Hospital. This study warrants the need for the judicious use of antibiotics as well as emphasize the use of modern diagnostic tools for the early detection of MDR and ESBL producers to curb the emergence and spread of MDR and ESBL producing bacteria in the clinical settings of Nepal.

Intestinal Parasitoses among Chepang and Musahar Community People of Makwanpur and Nawalparasi Districts of Nepal.

BACKGROUND: Intestinal parasitosis is one of the commonly perceived serious problems often observed in children leading to high mortality. The objective of the study was to identify the intestinal parasites and study their prevalence in the two mostly disadvantaged communities (Musahar and Chepang) of Nepal. METHODS: This was a cross-sectional study conducted in the Musahar and Chepang communities of Nepal from April to October 2019. A total of 205 random stool samples were collected in dry, clean and screw-capped plastic containers and mixed with 2.5% potassium dichromate solution. A pre-structured questionnaire was used to collect data on predisposing factors. The laboratory examination of the stool samples was done by direct microscopy and further confirmed by concentration methods (formalin ether sedimentation technique and flotation technique using Sheather's sugar solution), and modified acid-fast staining. Detection of eggs of Enterobius vermicularis was done by cellophane tape method. RESULTS: The overall prevalence of intestinal parasitic infection was found to be 36.6%, with a similar prevalence in the Chepangs (39.8%) and in the Musahars (33.3%) (P > 0.05). The most predominant helminth was Ascaris lumbricoides (15.6%), while the most prevalent protozoan was Entamoeba histolytica/dispar (5.4%). The study also assessed a significant association between the prevalence of parasites with socio-demographic factors, types of drinking water consumption and sanitation habits of the people (P < 0.05). CONCLUSION: The findings of the study suggest a need for formulating effective preventive and control strategies against intestinal parasitic infections along with the continuity of mass deworming program.

Wolbachia: a possible weapon for controlling dengue in Nepal.

Dengue, a mosquito-borne viral infectious disease, causes a high morbidity and mortality in tropical and subtropical areas of the world. In Nepal, the first case of dengue was reported in 2004 followed by frequent outbreaks in subsequent years, with the largest being in 2019 taking the death toll of six. It is reported that the number of dengue fever cases are soaring in Nepal spreading from the plains to more hilly regions. This might have serious public health implications in the future when combined with other factors, such as: global warming, lack of early detection and treatment of dengue, lack of diagnostic facilities, poor healthcare systems and mosquito control strategies. Nepal, thus, needs a cost-effective mosquito control strategy for the prevention and control of dengue. The Wolbachia-mediated biological method of the dengue control strategy is novel, economic, and environment-friendly. It has been successfully trialed in several areas of dengue-prone countries of the world, including Australia, Malaysia, Vietnam etc. resulting in significant reductions in dengue incidence. Given the lack of effective vector control strategy and weak economic condition of the country along with the persistence of climate and environment conditions that favors the host (Aedes mosquito) for Wolbachia, this approach can be a promising option to control dengue in Nepal.

Study on biodegradation kinetics of di-2-ethylhexyl phthalate by newly isolated halotolerant Ochrobactrum anthropi strain L1-W.

OBJECTIVE: Di-2-ethylhexyl phthalate (DEHP) pollution is one of the major environmental concerns all over the world. This research aimed at studying the biodegradation kinetics of DEHP by a newly isolated bacterial strain. Water and sediment samples were collected from Wuhan South Lake and potent bacterial isolates were screened for DEHP degradation, characterized by biochemical, physiological, morphological and 16S rDNA gene sequencing, and optimized under suitable pH, temperature, NaCl and DEHP concentrations. DEHP and its metabolites were quantified by High Performance Liquid Chromatography and their degradation kinetics were studied. RESULTS: The newly isolated bacterium was identified as Ochrobactrum anthropi strain L1-W with 99.63% similarity to Ochrobactrum anthropi ATCC 49188. It was capable of utilizing DEHP as the carbon source. The optimum growth temperature, pH, DEHP and NaCl concentration for the strain L1-W were 30 °C, 6, 400 mg/L and 10 g/L respectively. Strain L1-W was capable of degrading almost all (98.7%) of DEHP when the initial concentration was 200 mg/L within a period of 72 h. Besides, it was also found capable of degrading five other phthalates, thus making it a possible candidate for bioremediation of phthalates in the environmental settings.

Screening and Optimization of Newly Isolated Thermotolerant Lysinibacillus fusiformis Strain SK for Protease and Antifungal Activity.

The current study was designed to isolate, identify and characterize a Bacillus sp. capable of producing protease and exhibiting antifungal activity. A highly potent bacterium capable of producing protease abundantly was isolated from the soil collected from the waste pit near Microbiology Laboratory of Birendra Multiple Campus, Bharatpur and later on identified as Lysinibacillus fusiformis strain SK on the basis of morphological, physiological, biochemical and 16S rDNA gene sequencing techniques. The strain SK showed 98.36% similarity with L. fusiformis strain NBRC 15717. Using R-programming statistical analysis tool, the optimum incubation time for the highest average protease production (APP) (47.2 U/mL) was found to be 22 h at 50 °C and both incubation time and temperature showed a significant impact on the production of protease (P < 0.01). The maximum average relative protease activity (ARPA) was observed at pH 7.8 and 48 °C, whereas the least ARPA was observed in the presence of 80 g/L NaCl and 10 g/L HgCl2 (P < 0.01). The newly isolated bacterial strain also exhibited strong antifungal activity against aflatoxigenic Aspergillus flavus and Aspergillus parasiticus suggesting that it can be a potential candidate for protease production and activity over a wider range of temperature and pH as well as for synthesizing effective antifungal compounds.