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.

Extended spectrum ß-lactamase producing uropathogenic Escherichia coli and the correlation of biofilm with antibiotics resistance in Nepal.

BACKGROUND: Urinary tract infection (UTI) is one of the frequently diagnosed infectious diseases which is caused mainly by Escherichia coli. E. coli confers resistance against the two major classes of antibiotics due to the production of extended spectrum ß-lactamase enzymes (ESBL), biofilm, etc. Biofilm produced by uropathogenic E. coli (UPEC) protects from host immune system and prevent entry of antimicrobial compounds. The main objective of this cross-sectional study was to determine the correlation of biofilm production and antibiotic resistance as well as to characterize the pgaA and pgaC genes responsible for biofilm formation among uropathogenic ESBL producing E. coli. METHODS: A total of 1977 mid-stream urine samples were examined and cultured for bacterial strain identification. ESBL was detected by combined disc method following CLSI whereas biofilm formation was analyzed by semi-quantitative method. Furthermore, the pgaA and pgaC genes responsible for biofilm formation in UPEC were detected by multiplex PCR. All the statistical analyses were done via IBM SPSS Statistics 21 where Pearson's correlation test were used to determine correlation (-1 ≥ r ≤ 1). RESULTS: E. coli was the predominant causative agent, which accounted 159 (59.3%) of the Gram-negative bacteria, where 81 (50.9%) E. coli strains were found to be ESBL producers. In addition, 86 (54.1%) E. coli strains were found to be biofilm producers. Both the pgaA and pgaC genes were detected in 45 (93.7%) the UPEC isolates, which were both biofilm and ESBL producers. Moreover, there was a positive correlation between biofilm and ESBL production. CONCLUSION: The analyses presented weak positive correlation between biofilm and ESBL production in which biofilm producing UPEC harbors both pgaA and pgaC genes responsible for biofilm formation.

Inducible Clindamycin Resistance and Biofilm Production among Staphylococci Isolated from Tertiary Care Hospitals in Nepal.

Resistance to antibiotics, biofilm formation and the presence of virulence factors play important roles in increased mortality associated with infection by staphylococci. The macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat infections by methicillin-resistant isolates. Clinical failure of clindamycin therapy has been reported due to multiple mechanisms that confer resistance to MLSB. This study aims to find the incidence of different phenotypes of MLSB resistance and biofilm production among staphylococci. A total of 375 staphylococci were isolated from different clinical samples, received from two tertiary care hospitals in Nepal. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by D test, according to CLSI guidelines. Biofilm formation was detected by the tissue culture plate method and PCR was used to detect ica genes. Of the total staphylococci isolates, 161 (42.9%) were Staphylococcus aureus, with 131 (81.4%) methicillin-resistant strains, and 214 (57.1%) isolates were coagulase-negative staphylococci, with 143 (66.8%) methicillin-resistant strains. The overall prevalence of constitutive MLSB (cMLSB) and inducible MLSB (iMLSB) phenotypes was 77 (20.5%) and 87 (23.2%), respectively. Both iMLSB and cMLSB phenotypes predominated in methicillin-resistant isolates. The tissue culture plate method detected biofilm formation in 174 (46.4%) isolates and ica genes in 86 (22.9%) isolates. Among biofilm producing isolates, cMLSB and iMLSB phenotypes were 35 (20.1%) and 27 (15.5%), respectively. The cMLSB and iMLSB were 11 (12.8%) and 19 (22.1%), respectively, in isolates possessing ica genes. Clindamycin resistance in the form of cMLSB and iMLSB, especially among MRSA, emphasizes the need for routine D tests to be performed in the lab.

Preparatory phase for clinical trials of COVID-19 vaccine in Nepal.

Public health data suggested a rapid rise in COVID-19-confirmed cases in Nepal along with increased deaths. There has been a wide variation in clinical outcomes of this disease. Control of this pandemic depends on the availability of vaccines or drugs for SARS-CoV-2. Thus, viral and human genetics/genomics and immunology are necessary to understand whether these factors will affect clinical trials of vaccines in Nepal.

Glucose 6 phosphate dehydrogenase deficiency and hemoglobinopathy in South Western Region Nepal: a boon or burden.

OBJECTIVES: The study was carried out to optimize the phenotypic method to characterize the sickle cell trait (SCT), sickle cell anemia (SCA), and ß-thalassemia (ß-TT) suspected sample from tharu community of South Western province-5, Nepal. SCT and SCA were further evaluated by genotypic method employing amplification refractory mutation system (ARMS PCR). Moreover, Glucose 6 phosphate dehydrogenase (G6PD) was estimated in those hemoglobinopathy to observe its prevalence. The accurate and reliable method can play an important role in reduction of morbidity and mortality rate. RESULTS: The 100 suspected cases were subjected to phenotypic method adopting cellulose acetate electrophoresis and genotypic method using ARMS PCR which portraits (5%) SCA positive test showing HBS/HBS, (38%) SCT positive trait HBA/HBS and (36%) cases normal HBA/HBA. ß-TT (21%) cases were confirmed by electropherogram. G6PD deficiency was observed in (40%) of SCA, (18.4%) of SCT, (4.8%) of ß-TT and (2.8%) in normal cases. Increased G6PD were developed only in SCT (5.3%) and ß-TT (4.8%). The study highlighted sickle cell disorder (SCD) and ß-TT as the most common hemoglobinopathy coexisting with G6PD deficiency. Though hemoglobinopathy sometime could be protective in malaria but G6PD deficiency can cause massive hemolysis which may exacerbate the condition.

Susceptibility to Vancomycin of Biofilm Producing Staphylococci Isolated from Tertiary Care Hospital of Nepal / Susceptibilidad a la vancomicina de estafilococos productores de biopelículas aislados del hospital de atención terciaria de Nepal

Background: Methicillin resistance and biofilm-producing Staphylococci are emerging as multidrug-resistant strains narrowing the efficacy of antimicrobial therapy. Although vancomycin is used as the drug of choice to treat such isolates, different studies worldwide have documented the emergence of strains that are intermediately susceptible or resistant to this antibiotic. Objective: The study aimed to determine the minimum inhibitory concentration of vancomycin to methicillin-resistant and biofilm-producing staphylococci isolated from different clinical specimens. Methods: 375 staphylococci isolated from different clinical specimens over one year were included in the study. Biofilm formation was determined by the Tissue culture plate method (TCP), and ica genes were identified by Polymerase Chain Reaction (PCR). Antibiotic susceptibility and methicillin resistance were done following Clinical and Laboratory Standards Institute (CLSI) guidelines. The minimum inhibitory concentration (MIC) of vancomycin in all isolates was determined by the agar dilution method. Results:Among 375 Staphylococci studied, 43% and 57% represented S. aureus and Coagulase-Negative Staphylococci (CNS), respectively. The rate of Methicillin-Resistant S. aureus (MRSA) and Methicillin-Resistant Coagulase Negative Staphylococci (MRCNS) were 81.4% and 66.8% respectively and determined by the disc diffusion method. The most potential antibiotics were tetracycline and chloramphenicol showing sensitivity to more than 90% isolates. The Minimum Inhibitory Concentration (MIC) value of oxacillin for staphylococci ranged from 0.125-32 µg/ml. Oxacillin agar diffusion method showed 51.6% and 79.9% isolates as MRSA and MRCNS, respectively, revealing a very high percentage of S. aureus and CNS isolates as methicillin-resistant. All isolates had susceptible vancomycin MICs that ranged from 0.125-2 µg/ml. Two S. aureus isolated from Central Venous Catheter (CVC) and catheter specimens were detected with intermediate susceptibility to vancomycin. Similarly, three CNS isolated from blood, CVC, and wound/pus (w/p) were intermediately susceptible to vancomycin. Strong biofilm formation was observed in 22.1% of clinical isolates, and the ica gene was detected among 22.9% of isolates. Only one S. aureus detected as a biofilm producer by the TCP method was found to have intermediate susceptibility to vancomycin. Conclusions: The increment in vancomycin MIC among methicillin-resistant and biofilm-producing staphylococci is alarming. Strict control measures to prevent methicillin-resistant isolates spread and routine surveillance for vancomycin-resistant isolates must be incorporated in hospitals to prevent antimicrobial treatment failure

Antecedentes: Los estafilococos resistentes a la meticilina y productores de biopelículas están surgiendo como cepas multirresistentes que reducen la eficacia del tratamiento antimicrobiano. Aunque la vancomicina se utiliza como fármaco de elección para tratar dichos aislados, diferentes estudios realizados en todo el mundo han documentado la aparición de cepas intermedias susceptibles o resistentes a este antibiótico. Objetivo: El estudio tenía como objetivo determinar la concentración mínima inhibitoria de la vancomicina para los estafilococos resistentes a la meticilina y productores de biofilm aislados de diferentes muestras clínicas. Métodos: Se incluyeron en el estudio 375 estafilococos aislados de diferentes muestras clínicas durante un año. La formación de biopelículas se determinó mediante el método de la placa de cultivo de tejidos (TCP), y los genes ica se identificaron mediante la reacción en cadena de la polimerasa (PCR). La susceptibilidad a los antibióticos y la resistencia a la meticilina se realizaron siguiendo las directrices del Clinical and Laboratory Standards Institute (CLSI). La concentración inhibitoria mínima (MIC) de vancomicina en todos los aislados se determinó por el método de dilución en agar. Resultados:Entre los 375 estafilococos estudiados, el 43% y el 57% representaban S. aureus y estafilococos coagulasa-negativos (ECN), respectivamente. La tasa de S. aureus resistente a la meticilina (SARM) y de estafilococos coagulasa negativos resistentes a la meticilina (ECNM) fue del 81,4% y el 66,8%, respectivamente, y se determinó por el método de difusión de discos. Los antibióticos más potenciales fueron la tetraciclina y el cloranfenicol, que mostraron una sensibilidad superior al 90% de los aislados. El valor de la concentración inhibitoria mínima (CIM) de la oxacilina para los estafilococos osciló entre 0,125-32 µg/ml. El método de difusión en agar de la oxacilina mostró que el 51,6% y el 79,9% de los aislados eran SARM y MRCNS, respectivamente, lo que revela que un porcentaje muy elevado de los aislados de S. aureus y CNS son resistentes a la meticilina. Todos los aislados tenían MIC de vancomicina susceptibles que oscilaban entre 0,125-2 µg/ml. Se detectaron dos S. aureus aislados de muestras de catéteres venosos centrales (CVC) y catéteres con una susceptibilidad intermedia a la vancomicina. Del mismo modo, tres S. aureus aislados de sangre, CVC y herida/pus (w/p) fueron intermedianamente susceptibles a la vancomicina. Se observó una fuerte formación de biopelículas en el 22,1% de los aislados clínicos, y se detectó el gen ica en el 22,9% de los aislados. Sólo un S. aureus detectado como productor de biopelículas por el método TCP resultó tener una susceptibilidad intermedia a la vancomicina. Conclusiones: El incremento de la MIC de vancomicina entre los estafilococos resistentes a la meticilina y productores de biofilm es alarmante. Para evitar el fracaso del tratamiento antimicrobiano, deben incorporarse en los hospitales medidas de control estrictas para prevenir la propagación de los aislados resistentes a la meticilina y una vigilancia rutinaria de los aislados resistentes a la vancomicina

Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis.

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M. tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M. tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.