Biofilm and MBL production among imipenem resistant Pseudomonas aeruginosa and Acinetobacter species

Pseudomonas aeruginosa and Acinetobacter species are the primary cause of nosocomial infections. The advent of Metallo-beta-lactamase (MBL) and biofilm-producing bacterial strains poses a serious threat to reserve drugs such as carbapenem. The objective of this study was to determine the rate of MBL and biofilm production among imipenem resistant P. aeruginosa (IRPA) and imipenem resistant Acinetobacter spp. (IRAS) isolates. A total of 79 P. aeruginosa and 117 Acinetobacter spp. were isolated from various clinical specimens of patients from July 2016 to January 2017 at Manipal Teaching Hospital, Pokhara. MBL in IRPA and IRAS isolates were detected by Combined disc test and E-test. Biofilm production in imipenem resistant isolates was carried out by Microtitre plate assay. Fifteen (19%) P. aeruginosa and 57 (48.7%) Acinetobacter spp. were imipenem resistant isolates. MBL producers were found among 53.3% of IRPA and 38.6% of IRAS, whereas 100% of IRPA and 82.5% of IRAS were biofilm producers. All the biofilm producer IRPA isolates were Extensively Drug-Resistant (XDR), and a larger proportion of XDR IRAS strains were of high biofilm-producing phenotype. However, the majority of imipenem resistant (80% of IRPA and 49.1% of IRAS) and MBL producing (63%) isolates were weak biofilm formers. The study demonstrated the high capability of IRPA and IRAS to form a biofilm, which was strongly related to higher drug resistance. Nonetheless, imipenem resistant and MBL producer isolates showed an analogous association with the degree of biofilm formation. These MBL cum biofilm producer isolates were better susceptible to polymyxin B and ampicillin-sulbactam. DOI: http://dx.doi.org/10.5281/zenodo.419547

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

The durability of long-lasting insecticidal nets distributed to the households between 2009 and 2013 in Nepal

Background: Understanding and improving the durability of long-lasting insecticidal nets (LLINs) in the field are critical for planning future implementation strategies including behavioral change for care and maintenance. LLIN distribution at high coverage is considered to be one of the adjunctive transmission reduction strategies in Nepal’s Malaria Strategic Plan 2014–2025. The main objective of this study was to assess the durability through assessment of community usage, physical integrity, residual bio-efficacy, and chemical retention in LLINs: Interceptor®, Yorkool®, and PermaNet ®2.0 which were used in Nepal during 2009 through 2013. Methods: Assessments were conducted on random samples (n = 440) of LLINs from the eleven districts representing four ecological zones: Terai plain region (Kailali and Kanchanpur districts), outer Terai fluvial ecosystem (Surkhet, Dang, and Rupandhei districts), inner Terai forest ecosystem (Mahhothari, Dhanusa, and Illam districts), and Hills and river valley (Kavrepalanchock and Sindhupalchok districts). For each LLIN, fabric integrity in terms of proportionate hole index (pHI) and residual bio-efficacy were assessed. However, for chemical retention, a representative sample of 44 nets (15 Yorkool®, 10 Permanet®2.0, and 19 Interceptor®) was evaluated. Data were analyzed using descriptive statistics stratified by LLINs brand, districts, and duration of exposure. Results: On average, duration of use of LLINs was shortest for the Yorkool® samples, followed by PermaNet® 2.0 and Interceptor® with median ages of 8.9 (IQR = 0.4), 23.8 (IQR = 3.2), and 50.1 (IQR = 3.2) months, respectively. Over 80% of field distributed Yorkool® and PermaNet® 2.0 nets were in good condition (pHI< 25) compared to Interceptor® (66%). Bio-efficacy analysis showed that average mortality rates of Interceptor and Yorkool were below World Health Organization (WHO) optimal effectiveness of ≥ 80% compared to 2-year-old PermaNet 2.0 which attained 80%. Chemical retention analysis was consistent with bio-efficacy results. Conclusion: This study shows that distribution of LLINs is effective for malaria control; however, serviceable life of LLINs should be considered in terms of waning residual bio-efficacy that warrants replacement. As an adjunctive malaria control tool, National Malaria Control Program of Nepal can benefit by renewing the distribution of LLINs in an appropriate time frame in addition to utilizing durable and effective LLINs

Streptomycin – Like Antibiotic from Streptomyces spp. Isolated from Mount Everest

Streptomyces spp. (Lob18.2b), isolated from soil sample from Everest Base Camp, was obtained from Research Laboratory for Biotechnology and Biochemistry (RLABB). The isolate was found to inhibit Salmonella paratyphi, Salmonella typhi, Proteus mirabilis, Proteus vulgaris, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Bacillus subtilis and Staphylococcus aureus on primary screening. Secondary screening was done using fermented starch casein broth of the streptomycete to its stationary phase culture. The antibacterial agent was highly effective against all susceptible Gram negative bacteria except Proteus spp. Gram positive bacteria were relatively lesser sensitive. Pseudomonas aeruginosa was resistant to the agent. Antibacterial activity of aqueous fraction obtained from fermented broth of streptomycete culture was more effective than that of organic fraction of same extract. Thin layer chromatography revealed that the test compound was relatively nonpolar compared to the known antibiotics. Among the tested standard antibiotics, the chemical characteristic of the antibacterial agent was comparable to streptomycin

Renal impairment in stroke patients: A comparison between the haemorrhagic and ischemic variants [version 2; referees: 2 approved, 1 not approved]

Background: Renal impairment is regularly seen in hospitalized stroke patients, affecting the outcome of patients, as well as causing difficulties in their management. A prospective cohort study was conducted to assess the trend of renal function in hospitalized ischemic and haemorrhagic stroke patients. The incidence of renal impairment in these subgroups, the contributing factors and the need for renal replacement in renal impaired patients was evaluated. Methods: Alternate day renal function testing was performed in hospitalized stroke patients. Estimated glomerular filtration rate (e-GFR) was calculated and the trend of renal function in the two stroke subgroups (haemorrhagic and ischemic) was assessed, with renal impairment defined as e-GFR < 60mL/ minute per 1.73m2. Results: Among 52 patients, 25 had haemorrhagic stroke (mean age 59.81 ± 14.67) and 27 had ischemic stroke (mean age 56.12 ± 13.08). The mean e-GFR (mL/minute per 1.732m2) at admission in the haemorrhagic stroke subgroup was 64.79 ± 25.85 compared to 86.04 ± 26.09 in the ischemic stroke subgroup (p=0.005). Sixteen out of 25 (64%) patients in the haemorrhagic stroke subgroup and 9 out of 27 (33.3%) patients in the ischemic subgroup developed renal impairment (p=0.027). The location of the bleed (p=0.8), volume of hematoma (p=0.966) and surgical intervention (p=0.4) did not predispose the patients to renal impairment. One out of 16 patients with haemorrhagic stroke (who eventually died), and 2 out of 9 patients with ischemic stroke required renal replacement. Conclusion: Renal impairment is commonly seen in stroke patients, more so in patients who suffered haemorrhagic strokes.  The impairment, however, is transient and rarely requires renal replacement therapy

Renal impairment in stroke patients: A comparison between the haemorrhagic and ischemic variants [version 1; referees: 2 approved, 1 not approved]

Background: Renal impairment is regularly seen in hospitalized stroke patients, affecting the outcome of patients, as well as causing difficulties in their management. A prospective cohort study was conducted to assess the trend of renal function in hospitalized ischemic and haemorrhagic stroke patients. The incidence of renal impairment in these subgroups, the contributing factors and the need for renal replacement in renal impaired patients was evaluated. Methods: Alternate day renal function testing was performed in hospitalized stroke patients. Estimated glomerular filtration rate (e-GFR) was calculated and the trend of renal function in the two stroke subgroups (haemorrhagic and ischemic) was assessed, with renal impairment defined as e-GFR < 60mL/ minute per 1.73m2. Results: Among 52 patients, 25 had haemorrhagic stroke (mean age 59.81 ± 14.67) and 27 had ischemic stroke (mean age 56.12 ± 13.08). The mean e-GFR (mL/minute per 1.732m2) at admission in the haemorrhagic stroke subgroup was 64.79 ± 25.85 compared to 86.04 ± 26.09 in the ischemic stroke subgroup (p=0.005). Sixteen out of 25 (64%) patients in the haemorrhagic stroke subgroup and 9 out of 27 (33.3%) patients in the ischemic subgroup developed renal impairment (p=0.27). The location of the bleed (p=0.8), volume of hematoma (p=0.966) and surgical intervention (p=0.4) did not predispose the patients to renal impairment. One out of 16 patients with haemorrhagic stroke (who eventually died), and 2 out of 9 patients with ischemic stroke required renal replacement. Conclusion: Renal impairment is commonly seen in stroke patients, more so in patients who suffered haemorrhagic strokes.  The impairment, however, is transient and rarely requires renal replacement therapy

Growing Resistance to Vancomycin among Methicillin Resistant Staphylococcus Aureus Isolates from Different Clinical Samples

Introduction: Methicillin resistant Staphylococcus aureus (MRSA), majorly associated with nosocomial and community infections worldwide, are emerging as resistant strains to many antibiotics narrowing down the efficacy of antimicrobial therapy. In order to investigate the changing resistant pattern of MRSA to empirical drugs, the study was carried out at KIST Medical College and Hospital, Nepal. It also aims to determine the minimum inhibitory concentration of vancomycin among MRSA. &#13; Methods: Altogether 3500 clinical samples including 1303 blood, 1489 urine and 708 body fluids were collected and processed. Isolated S. aureus were further screened for methicillin resistance by Kirby-Bauer disk diffusion technique using cefoxitin (30μg) disk. All MRSA were subjected to in vitro determination of MIC of vancomycin by agar dilution method as recommended by CLSI guidelines. &#13; Results: Total 287 S. aureus were isolated from the different clinical samples. Altogether 248 (86.41%) were found to be multidrug resistance (MDR) while 42 (14.63%) of the isolates were methicillin resistance with the highest prevalence in the age group of 16-30. All 42 (100%) MRSA isolates were resistant to ampicillin and penicillin followed by 41 (97.62%), 32 (76.19%), 31(73.81%), 29 (69.05%), 9 (21.43%) and seven (16.67%) to cefotaxime, gentamycin, cotrimoxazole, erythromycin, tetracycline and ciprofloxacin respectively. Although all MRSA strains were sensitive to vancomycin on disc diffusion, four isolates were intermediates in vitro determination of MIC of vancomycin. The break point for vancomycin was found to be 15mm. &#13; Conclusions: The increment in vancomycin MIC among MRSA is alarming. Strict control measures to prevent MRSA spread and a routine surveillance for VRSA must be incorporated in hospitals. &#13; Keywords: mdr; mrsa; mic; visa; vrsa

Growing Resistance to Vancomycin among Methicillin Resistant Staphylococcus Aureus Isolates from Different Clinical Samples

Introduction: Methicillin resistant Staphylococcus aureus (MRSA), majorly associated with nosocomial and community infections worldwide, are emerging as resistant strains to many antibiotics narrowing down the efficacy of antimicrobial therapy. In order to investigate the changing resistant pattern of MRSA to empirical drugs, the study was carried out at KIST Medical College and Hospital, Nepal. It also aims to determine the minimum inhibitory concentration of vancomycin among MRSA. Methods: Altogether 3500 clinical samples including 1303 blood, 1489 urine and 708 body fluids were collected and processed. Isolated S. aureus were further screened for methicillin resistance by Kirby-Bauer disk diffusion technique using cefoxitin (30μg) disk. All MRSA were subjected to in vitro determination of MIC of vancomycin by agar dilution method as recommended by CLSI guidelines. Results: Total 287 S. aureus were isolated from the different clinical samples. Altogether 248 (86.41%) were found to be multidrug resistance (MDR) while 42 (14.63%) of the isolates were methicillin resistance with the highest prevalence in the age group of 16-30. All 42 (100%) MRSA isolates were resistant to ampicillin and penicillin followed by 41 (97.62%), 32 (76.19%), 31(73.81%), 29 (69.05%), 9 (21.43%) and seven (16.67%) to cefotaxime, gentamycin, cotrimoxazole, erythromycin, tetracycline and ciprofloxacin respectively. Although all MRSA strains were sensitive to vancomycin on disc diffusion, four isolates were intermediates in vitro determination of MIC of vancomycin. The break point for vancomycin was found to be 15mm. Conclusions: The increment in vancomycin MIC among MRSA is alarming. Strict control measures to prevent MRSA spread and a routine surveillance for VRSA must be incorporated in hospitals.  Keywords: mdr; mrsa; mic; visa; vrsa

Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain

Random Amplified Polymorphic DNA (RAPD) is a method of producing a genetic fingerprint of a particular species without its prior genetic information. Relationship between species may be determined by comparing their unique fingerprint information. B. thuringiensis was isolated from soil samples of Khumbu base camp of Everest region, Nepal. Crystal protein (delta endotoxin) producing strains (46 from Phereche and 40 from Sagarmatha national park) were tested against a series of 100 decamer RAPD primers (codes 201-300, obtained from University of British Columbia) by RAPD PCR. Primer 284 was found the best among the tested primers and the reaction condition for PCR was optimized with a PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl 2 with pH 8.3.; 200ìm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1 % DMSO as a final concentrations in 25ìl reaction mixture. The thermal programme was programmed as initial denaturation temperature at 94 o C for 5 min followe

Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIV/AIDS in Nepal

BACKGROUND: Bacterial opportunistic infections are common in people living with HIV/AIDS (PLHA). Besides HIV-TB co-infection, lower respiratory tract infections (LRTIs) due to multidrug-resistant (MDR) bacteria cause significant morbidity and mortality among PLHA. This study identified bacterial co-infection of the lower respiratory tract and detected plasmid-mediated blaTEM and blaCTX-M genes among Extended-Spectrum β-Lactamase (ESBL) producing isolates from sputum samples in PLHA. METHODS: A total of 263 PLHA with LRTIs were enrolled in this study, out of which, 50 were smokers, 70 had previous pulmonary tuberculosis, and 21 had CD4 count &lt; 200 cells/µl. Sputum samples collected from PLHA were processed with standard microbiological methods to identify the possible bacterial pathogens. The identified bacterial isolates were assessed for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method following Clinical Laboratory Standard Institute (CLSI) guidelines. In addition, plasmid DNA was extracted from MDR and ESBL producers for screening of ESBL genes; blaCTX-M and blaTEM by conventional PCR method using specific primers. RESULTS: Of 263 sputum samples, 67 (25.48%) showed bacterial growth. Among different bacterial pathogens, Klebsiella pneumoniae, (17; 25.37%) was the most predominant, followed by Haemophillus influenzae, (14; 20.90%) and Escherichia coli, (12; 17.91%). A higher infection rate (4/8; 50%) was observed among people aged 61–70 years, whereas no infection was observed below 20 years. About 30.0% (15/50) of smokers, 32.86% (23/70) cases with previous pulmonary tuberculosis, and 52.38% (11/21) with CD4 count &lt; 200 cells/µl had bacterial LRTIs. Among 53 bacterial isolates excluding H. influenzae, 28 isolates were MDR and 23 were ESBL producers. All ESBL producers were sensitive to colistin and polymyxin B. Among ESBL producers, 47.83% (11/23) possessed blaCTX-M, 8.6% (2/23) were positive for blaTEM gene, and 43.48% (10/23) possessed both ESBL genes. CONCLUSION: The increasing rate of MDR bacterial infections, mainly ESBL producers of LRTIs causes difficulty in disease management, leading to high morbidity and mortality of PLHA. Hence, it is crucial to know the antibiogram pattern of the isolates to recommend effective antimicrobial therapy to treat LRTIs in PLHA