Emergence of mcr-1 Gene in Colistin-Resistant Escherichia coli Isolates from Chicken in Chitwan, Nepal.

The escalating prevalence of colistin-resistant Escherichia coli in poultry has emerged as a significant concern. This study aimed to assess the occurrence of the mcr-1 gene in colistin-resistant E. coli isolates from poultry samples. A cross-sectional study was conducted at National Avian Disease Investigation Laboratory, Nepal, on 210 chicken meat samples, including liver, heart, and spleen. E. coli was isolated and identified by conventional cultural methods. Antibiotic resistance pattern was assessed by the Kirby-Bauer disc diffusion method. The mcr-1 gene was detected by conventional polymerase chain reaction. The average viable count in chicken meat samples was log 6.01 CFU (colony-forming unit)/g, whereas the average coliform count was log 3.85 CFU/g. Coliforms were detected in at least one sample from 48.01% of total samples. The prevalence of E. coli in all meat samples was 39.52%. Liver accounted for the largest fraction of E. coli isolates (45.45%). Cefepime was the most effective antibiotic. Among all isolates, 45 (54.21%) were multidrug-resistant E. coli, 17 (20.48%) were colistin-resistant E. coli, and 11 (64.70%) harbored the mcr-1 gene. High prevalence of multidrug-resistant E. coli isolates, colistin-resistant isolates, and mcr-1 gene-carrying isolates indicates a serious concern, as it could potentially lead to colistin resistance in human pathogens through horizontal transfer of resistant genes from poultry to humans.

Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing.

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test's single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn't differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn't differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1-0.4]; Method 3: 0.3U/gHb [IQR: 0.1-0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2-0.6]; Method 3: 0.3U/gHb [IQR: 0.1-0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn't improve repeatability. Delays of up to 5 hours didn't result in a clinically relevant difference in measured G6PD activity. The manufacturer's recommended cut-off for intermediate deficiency is conservative.

Use of antimicrobials during the COVID-19 pandemic: A qualitative study among stakeholders in Nepal.

The COVID-19 pandemic was a major public health threat and the pressure to find curative therapies was tremendous. Particularly in the early critical phase of the pandemic, a lot of empirical treatments, including antimicrobials, were recommended. Drawing on interviews with patients, clinicians and drug dispensers, this article explores the use of antimicrobials for the management of COVID-19 in Nepal. A total of 30 stakeholders (10 clinicians, 10 dispensers and 10 COVID-19 patients) were identified purposively and were approached for an interview. Clinicians and dispensers in three tertiary hospitals in Kathmandu assisted in the recruitment of COVID-19 patients who were undergoing follow-up at an out-patient department. Interviews were audio recorded, translated and transcribed into English, and were analyzed thematically. The respondents report that over-the-counter (OTC) use of antibiotics was widespread during the COVID-19 pandemic in Nepal. This was mostly rooted in patients' attempts to mitigate the potential severity of respiratory illnesses, and the fear of the stigmatization and social isolation linked to being identified as a COVID-19 patient. Patients who visited drug shops and physicians reportedly requested specific medicines including antibiotics. Clinicians reported uncertainty when treating COVID-19 cases that added pressure to prescribe antimicrobials. Respondents from all stakeholder groups recognized the dangers of excessive use of antimicrobials, with some referring to the development of resistance. The COVID-19 pandemic added pressure to prescribe, dispense and overuse antimicrobials, accentuating the pre-existing OTC use of antimicrobials. Infectious disease outbreaks and epidemics warrant special caution regarding the use of antimicrobials and specific policy response.

Multidrug resistance, biofilm formation and detection of bla CTX-M and bla VIM genes in E. coli and Salmonella isolates from chutney served at the street-food stalls of Bharatpur, Nepal.

Antimicrobial resistance (AMR) amid the bacteria found in ready-to-eat foods is a grave concern today warranting an immediate intervention. The current study was undertaken to explore the status of AMR in E. coli and Salmonella species in ready-to-eat Chutney samples (n = 150) served at the street food stalls in Bharatpur, Nepal, with a major focus on detecting extended-spectrum ß-lactamase (ESBL) and metallo ß-lactamase (MBL) genes along with biofilm formation. Average viable counts, coliform counts, and Salmonella Shigella counts were 1.33 × 106±141481.4, 1.83 × 105±91303.6, and 1.24 × 105±63933.19 respectively. Out of 150 samples, 41 (27.33%) harbored E. coli, of which 7 were E. coli O157:H7; whereas Salmonella spp. were found in 31 (20.67%) samples. Bacterial contamination of Chutney by E. coli and Salmonella and ESBL-production were both found significantly affected by different sources of water used, personal hygiene and literacy rate of the vendors as well as by the type of cleaning materials used to wash knives and chopping boards (P < 0.05). Antibiotic susceptibility testing revealed that imipenem was the most effective drug against both types of bacterial isolates. Additionally, 14 (45.16%) Salmonella isolates and 27 (65.85%) E. coli isolates were found to be multi-drug resistant (MDR). Total ESBL (bla CTX-M) producers reported were 4 (12.90%) Salmonella spp. and 9 (21.95%) E. coli. Only 1 (3.23%) Salmonella spp. and 2 (4.88%) E. coli isolates were bla VIM gene carriers. Dissemination of knowledge of personal hygiene amongst the street vendors and consumer awareness regarding ready-to-eat foods are crucial factors that can be suggested to curtail the emergence and transmission of food-borne pathogens.

Bacterial Assessment of Stethoscopes Used by Healthcare Workers at a Tertiary Care Government Hospital in Bharatpur, Nepal.

The transmission of healthcare-associated infections (HCAIs) in healthcare settings is a serious challenge in the medical fraternity. Medical devices, such as stethoscopes used by healthcare workers (HCWs), are likely to harbor a considerable number of pathogenic microbes, which may result in the transmission of HCAIs. This study sought to investigate bacterial contamination of stethoscopes used by HCWs at Bharatpur Hospital, Nepal. During the study period of 3 months from December 2019 to February 2020, a total of 87 stethoscopes were examined; bacterial pathogens were isolated and identified by culture and biochemical tests, and their susceptibilities against different antibiotics were determined using standard protocols of the Clinical and Laboratory Standards Institute (CLSI). The disc diffusion method was used primarily to screen for extended-spectrum beta-lactamase (ESBL)- and metallo-beta-lactamase (MBL)-producing isolates, followed by their confirmation using cephalosporin/clavulanate combination discs and the disc potentiation methods, respectively. In addition, molecular detection of blaCTX-M and blaVIM genes was performed using conventional polymerase chain reaction (PCR). Of the 87 stethoscopes examined, more than a quarter (28.7%) were colonized with different pathogenic bacteria. Bacterial contamination of stethoscopes was found to be significantly associated with various factors, such as disinfecting routine, method of disinfection, and department of the hospital (p < 0.05). A higher rate of bacterial contamination was observed on the diaphragm of the stethoscope (12.64%) and among HCWs who overlooked hand hygiene practices (45.45%). The prevalence of methicillin-resistant S. aureus (MRSA) was 44.44%, and approximately half of the Gram-negative isolates (47%) were multidrug resistant (MDR). Imipenem (81.25%) and chloramphenicol (83.33%) were found to be the most effective antibiotics for Gram-negative and Gram-positive bacteria, respectively. Phenotypic screening showed that 43.75% of isolates were ESBL producers, and 18.75% were MBL producers, but blaCTX-M and blaVIM genes were detected in only 31.25% and 6.25% of isolates, respectively. The results of the study call for effective stethoscope disinfection practices along with the judicious use of antibiotics by HCWs in order to minimize cross-contamination, emergence of resistance, and spread of nosocomial infections in clinical settings.

Beta-Lactamase-Producing Gram-Negative Bacterial Isolates Among the Patients Attending a Tertiary Care Hospital, Kathmandu, Nepal.

Over the times, carbapenems have been the choice of drug for treating multidrug-resistant (MDR) and extended spectrum beta-lactamase (ESBL)-producing organisms. The current study aimed at determining the occurrence of metallo beta-lactamase (MBL) and AmpC beta-lactamase (ABL) in gram negative bacteria isolated from clinical samples. A cross-sectional study was conducted amongst the patients visiting Manmohan Memorial Medical College and Teaching Hospital (MMTH), Kathmandu, Nepal from August 2017 to January 2018. A total of 4351 samples including urine, pus, wound swab, endotracheal tip, catheter tip, and blood were collected from the patients and processed by standard conventional microbiological methods. Antibiotic susceptibility testing (AST) of the isolates was performed by Kirby-Bauer disk diffusion method. Double disc synergy test was performed on carbapenem resistant organisms to detect production of MBL and inhibitor-based test was used for the detection of ABL production. Of the 4351 samples, 421 bacterial isolates belonging to 16 different genera were recovered, of which 303 (71.97%) were Gram negative bacilli (GNB). E. coli (189/303) and S. aureus (80/118) were the most prevalent among gram negatives and gram positives, respectively. Bacterial incidence was found significantly associated with gender, specimen type, and the department where the patients were enrolled. Colistin-sulfate and polymycin-B were the most effective drug against GNB, whereas imipenem against gram positives. Prevalence of MDR and methicillin-resistant S. aureus (MRSA) was 35.15% and 60%, respectively. The prevalence of MBL and ABL-producing isolate was 11(3.6%) and 13(4.3%), respectively. Pseudomonas aeruginosa (5/11) and E. coli (9/13) were the major MBL and ABL producers, respectively. MBL and ABL production was found to be significantly associated with the age of the patient and the specimen type. A regular antibiotic surveillance activity with screening for MBL and ABL-producing bacterial isolates in the hospital settings to curb the incidence and transmission of such difficult-to-treat pathogens.

Evaluation of Xpert MTB/RIF Assay, MTB Culture and Line Probe Assay for the Detection of MDR Tuberculosis in AFB Smear Negative Specimens.

The global burden of tuberculosis (TB), particularly with multidrug resistance (MDR), is escalating and has become a major health challenge. It is well known that acid-fast bacilli (AFB) smear-negative TB patients are the major source of spreading TB to healthy individuals when left untreated. Early diagnosis of TB and rapid detection of drug resistance are important for the proper management of drug-resistant TB (DR-TB). Therefore, a laboratory based cross-sectional study was conducted from July to December 2019 at the National Tuberculosis Centre, Thimi, Nepal, with the objective of evaluating the diagnostic performance of Xpert MTB/RIF assay, Mycobacterium tuberculosis (MTB) culture and line probe assay (LPA) for the detection of MDR-TB in AFB smear-negative sputum samples. We evaluated a total of 222 AFB smear-negative sputum specimens, of which 21.6% (n = 48) showed MTB positive with Xpert MTB/RIF assay and, while culturing on Lowenstein-Jensen (LJ) media, 21.2% (n = 47) were MTB culture positive. The sensitivity, specificity, PPV and NPV at 95% confidence interval of Xpert MTB/RIF assay on diagnosing M. tuberculosis from smear-negative specimens were 73% (57-84), 92% (87-96), 71% (59-81) and 93% (89-95), respectively. In addition, the sensitivity of Xpert MTB/RIF assay and LPA in detecting rifampicin resistance was 75% (42-94, 95% CI) and 91.67% (62-99, 95% CI), respectively. The current study also assessed a significant association between the occurrence of pulmonary tuberculosis with different age group, TB history and alcohol consumption. These findings indicate that Xpert MTB/RIF assay and LPA are appropriate methods for early detection and accurate diagnosis of TB and RIF mono-resistant cases.

Detection of NDM Variants (bla NDM-1, bla NDM-2, bla NDM-3) from Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae: First Report from Nepal.

Background: Increasing burden of carbapenem resistance among Enterobacterales is attributable to their ability to produce carbapenemase enzymes like metallo-beta-lactamase (MBL), Klebsiella pneumoniae carbapenemase (KPC), and OXA-type. This study aimed to determine the prevalence of carbapenemases and MBL genes ((bla NDM-1, bla NDM-1 and bla NDM-3) among E. coli and K. pneumoniae isolates. Methods: A total of 2474 urine samples collected during the study period (July-December 2017) were processed at the microbiology laboratory of Kathmandu Model Hospital, Kathmandu. Isolates of E. coli and K. pneumoniae were processed for antimicrobial susceptibility testing (AST) by disc diffusion method. Carbapenem-resistant isolates were subjected to Modified Hodge Test (MHT) for phenotypic confirmation, and inhibitor-based combined disc tests for the differentiation of carbapenemase (MBL and KPC). MBL-producing isolates were screened for NDM genes by polymerase chain reaction (PCR). Results: Of the total urine samples processed, 19.5% (483/2474) showed the bacterial growth. E. coli (72.6%; 351/483) was the predominant isolate followed by K. pneumoniae (12.6%; 61/483). In AST, 4.4% (18/412) isolates of E. coli (15/351) and K. pneumonia (3/61) showed resistance towards carbapenems, while 1.7% (7/412) of the isolates was confirmed as carbapenem-resistant in MHT. In this study, all (3/3) the isolates of K. pneumoniae were KPC-producers, whereas 66.7% (10/15), 20% (3/15) and 13.3% (2/15) of the E. coli isolates were MBL, KPC and MBL/KPC (both)-producers, respectively. In PCR assay, 80% (8/10), 90% (9/10) and 100% (10/10) of the isolates were positive for bla NDM-1, bla NDM-2 and bla NDM-3, respectively. Conclusion: Presence of NDM genes among carbapenemase-producing isolates is indicative of potential spread of drug-resistant variants. This study recommends the implementation of molecular diagnostic facilities in clinical settings for proper infection control, which can optimize the treatment therapies, and curb the emergence and spread of drug-resistant pathogens.

Antibiotic Susceptibility, Biofilm Production, and Detection of mecA Gene among Staphylococcus aureus Isolates from Different Clinical Specimens.

The increasing incidence of methicillin-resistant and biofilm-forming S. aureus isolates in hospital settings is a gruesome concern today. The main objectives of this study were to determine the burden of S. aureus in clinical samples, assess their antibiotic susceptibility pattern and detect biofilm formation and mecA gene in them. A total of 1968 different clinical specimens were processed to isolate S. aureus following standard microbiological procedures. Antibiotic susceptibility test of the isolates was performed by Kirby-Bauer disc-diffusion method following CLSI guidelines. Biofilm was detected through tissue culture plate method. Methicillin-resistant S. aureus (MRSA) isolates were screened using cefoxitin (30 µg) discs and mecA gene was amplified by conventional polymerase chain reaction (PCR). Of 177 bacterial growth, the prevalence of S. aureus was 15.3% (n = 27). MRSA were 55.6% (15/27) and 44% (12/27) exhibited multidrug resistance (MDR). There was no significant association between methicillin resistance and MDR (p > 0.05). Both MRSA and MSSA were least sensitive to penicillin (100%, 75%) followed by erythromycin (86.6%, 66.6%). Most of the MRSA (93.4%) were susceptible to tetracycline. All S. aureus isolates were biofilm producers-19 (70%) were weak and only one (4%) was a strong biofilm producer. The strong biofilm-producing MSSA was resistant to most of the antibiotics except cefoxitin and clindamycin. None of the MSSA possessed mecA gene while 8 (53.3%) MRSA had it. More than half of S. aureus isolated were MRSA. High incidence of multidrug resistance along with capacity to form biofilm among clinical isolates of S.aureus is a matter of apprehension and prompt adoption of biosafety measures is suggested to curb their dissemination in the hospital environments.

Efficacy of Urine Dipstick Test in Diagnosing Urinary Tract Infection and Detection of the blaCTX-M Gene among ESBL-Producing Escherichia coli.

A urine dipstick test used for prompt diagnosis of urinary tract infection (UTI) is a rapid and cost-effective method. The main objective of this study was to compare the efficacy of the urine dipstick test with culture methods in screening for UTIs along with the detection of the blaCTX-M gene in extended spectrum ß-lactamase (ESBL)-producing Escherichia coli. A total of 217 mid-stream urine samples were collected from UTI-suspected patients attending Bharatpur Hospital, Chitwan, and tested by dipstick test strip (COMBI-10SL, Germany) prior to the culture. E. coli isolates were identified by standard microbiological procedures and subjected to antimicrobial susceptibility testing by Kirby Bauer disc diffusion method following CLSI guideline. Primary screening of ESBL-producing E. coli isolates was conducted using ceftriaxone, cefotaxime and ceftazidime discs and phenotypically confirmed by combined disk diffusion test. Plasmid DNA of ESBL-producing strains was extracted by phenol-chloroform method and subjected to PCR for detection of the blaCTX-M gene. Out of 217 urine samples, 48 (22.12%) showed significant bacteriuria. Among 46 (21.20%) Gram negative bacteria recovered, the predominant one was E. coli 37 (77.08%) of which 33 (89.19%) were multidrug resistant (MDR). E. coli isolates showed a higher degree of resistance towards cefazolin (62.16%) while 81.08% of the isolates were sensitive towards amikacin followed by nitrofurantoin (70.27%). Among 14 (37.84%) phenotypically confirmed ESBL isolates, only eight (21.62%) isolates carried the blaCTX-M gene. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of urine dipstick test were 43.75%, 77.51%, 35.59% and 82.91%, respectively. Besides, the use of dipstick test strip for screening UTI was associated with many false positive and negative results as compared to the gold standard culture method. Hence, dipstick nitrite test alone should not be used as sole method for screening UTIs.

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.

Bacterial etiology of sputum from tuberculosis suspected patients and antibiogram of the isolates.

OBJECTIVE: The current study aims to explore the bacteriology of sputum of tuberculosis (TB) suspected patients. A cross-sectional study was carried out in the sputum samples of 150 TB suspected patients visiting District Public Health Office, Bharatpur, Nepal. The samples were subjected to cultural, microscopic and biochemical analyses for the identification of the isolates. In addition, antibiotic susceptibility tests were carried out with a special focus on ESBL and MBL production following Clinical and Laboratory Standard Institute guidelines. RESULTS: Bacterial growth was recovered in 47% (71/150) of the TB suspected patients of which 12.66% (19/150) had pulmonary TB infection. Streptococcus spp. (9%) and Pseudomonas aeruginosa (9%) were the most frequently isolated bacteria. Enterobacteriaceae accounted for 35% of the total isolates. Occurrence of bacterial pathogens was more in males (69%) than in females (31%).The incidence of bacterial pathogen was seen associated with gender of the patients and with the TB infection (p < 0.05) but independent with age of the patients and HIV infection (p > 0.05). Tetracycline was effective against Streptococcus spp. whereas gentamicin was effective against Bacillus species. Imipenem and co-trimoxazole were effective drugs for Gram-negative isolates. Among 83 isolates, 35 were multi-drug resistant, 9 were ESBL producers and 4 were MBL producers.

Bacterial Etiology of Wound Exudates in Tertiary Care Cancer Patients and Antibiogram of the Isolates.

INTRODUCTION: Patients with malignancies frequently develop infections as a result of surgical procedures and fungating wounds leading to pus formation. This cross-sectional study was conducted to explore the bacteriological spectra of infections of various cancer sites and their antibiotic sensitivity patterns among the patients visiting minor operation theatre (OT) of B.P. Koirala Memorial Cancer Hospital (BPKMCH), Chitwan, Nepal. METHODS: Over a period of 3 months from September to November 2018, a total of 183 wound exudates and pus samples were collected and analyzed by standard microbiological procedures. Isolates were identified based on the colony characters, Gram staining and an array of biochemical tests. Antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion technique according to criteria set by CLSI, 2016. Methicillin resistance in Staphylococcus aureus was tested with the help of cefoxitin using disc diffusion method. RESULTS: Out of the 183 samples, 149 (81.4%) were culture positive. Among 13 different isolates identified, S. aureus (43.0%) was predominant followed by E. coli (14.0%). Higher incidence of bacteria was seen among the males (52.3%), in the age group 51 to 60 years (26.8%) and among the patients undergoing surgical intervention to deal with cancer (34.2%). The prevalence of wound infection was significantly affected by gender, age, and treatment regimen (P < .01). Out of the total 68 S. aureus isolates, 38 (44.1%) were deemed as Methicillin-resistant Staphylococcus aureus (MRSA). Among the 158 isolates, 85 (53.8%) were multi-drug resistant (MDR). Cefepime was the most effective antibiotic for Gram positive isolates whereas both imipenem and meropenem were found to be equally more effective for Gram negative isolates. CONCLUSION: This study suggests that patients with malignancies harbor pathogenic bacteria; therefore, prudent use of antibiotics is essential to prevent the emergence of MDR pathogens.

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.

Microbial Diversity and Antibiotic Susceptibility Pattern of Bacteria Associated with Motorcycle Helmets.

BACKGROUND: Motorcycle helmets can serve as a potential vehicle for the transmission of pathogenic bacteria and fungi with serious health implications. The main aim of this study was to explore the microbial diversity associated with the motorcycle helmets and determine the antibiotic susceptibility profile of the bacterial isolates. METHODS: A descriptive cross-sectional study was carried out among the teaching staffs of Birendra Multiple Campus, Bharatpur, Nepal. A total of 130 motorcycle helmets worn by the teaching staffs of the Birendra Multiple Campus, Bharatpur, were included in the study for microbiological investigations. RESULTS: Of the total 130 motorcycle helmets analyzed, 392 bacteria and 346 fungi belonging to seven different genera were recovered. Staphylococcus aureus 89 (22.7%) was the predominant bacteria followed by S. epidermidis 77 (19.6%) and E. coli 54 (13.8%), whereas Aspergillus niger 67 (19.4%) was the predominant fungi followed by A. fumigatus 49 (14.2%). Antibiotic susceptibility test was performed by the disc diffusion method for all the bacterial isolates. Tetracycline, gentamycin, and cotrimoxazole were the most effective antibiotics for Gram-positive isolates, whereas Gram-negative isolates were sensitive towards imipenem and ciprofloxacin. Of the total bacterial isolates, 153 (39.0%) were multidrug-resistant (MDR), 10.4% were extended-spectrum beta-lactamase (ESBL) producers, and 4.3% were metallo-beta-lactamase (MBL) producers and, out of 89 isolates of Staphylococcus aureus, 30 (33.7%) were detected as methicillin-resistant Staphylococcus aureus (MRSA). CONCLUSION: The findings suggest that motorcycle riders should follow good hygiene practices and regularly clean their helmets with suitable sterilants to avoid the risk of microbial contamination and reduce the associated risks.

Multi-drug resistant extended-spectrum beta-lactamase producing E. coli and Salmonella on raw vegetable salads served at hotels and restaurants in Bharatpur, Nepal.

OBJECTIVE: Antimicrobial resistance among the bacteria present in ready-to-eat foods like vegetable salads is an emerging concern today. The current study was undertaken to investigate the presence of multi-drug resistant extended-spectrum ß-lactamase (ESBL) producing E. coli and Salmonella spp. in raw vegetable salads served at hotels and restaurants in Bharatpur. A total of 216 salad samples were collected from three different grades of hotels and restaurants and examined for the presence of E. coli and Salmonella spp. in Microbiology laboratory of Birendra Multiple Campus by conventional microbiological techniques. RESULTS: Out of 216 samples, 66 samples (35.2%) showed the presence of Salmonella spp. whereas E. coli was recovered from 29 (13.4%) samples of which 3 samples harbored E. coli O157: H7. Antibiotic susceptibility testing revealed that 9 (13.6%) Salmonella and 4 (13.8%) E. coli isolates were detected as multi-drug resistant. Total ESBL producers reported were 5 (7.57%) Salmonella and 4 (13.8%) E. coli. The study also assessed a significant association between occurrence of E. coli and Salmonella with different grades of hotels and restaurants, personal hygiene and literacy rate of chefs and with the type of cleaning materials used to wash knives and chopping boards (p < 0.05). The findings suggest an immediate need of attention by the concerned authorities to prevent the emergence and transmission of food-borne pathogens and infections antimicrobial resistance among them.