Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Luminance contrast preferences of people with a vision impairment for elements in the built environment.

BACKGROUND: Standards for building elements recommend a minimum luminance contrast of 30%. The basis of this value and the metric originally used is not known. OBJECTIVE: To begin to provide an evidence base for the specification of minimum contrast in building elements. METHODS: Subjects with and without a vision impairment were characterized by visual acuity, contrast sensitivity, visual fields and color vision. On an iPad they rated the visibility, as a function of contrast, of simulated door frames, door handles, light switches and stair nosings as "not visible at all", "poorly visible", "easily visible" and "extremely easily visible". RESULTS: The contrasts for each level of visibility were highly correlated with visual acuity and contrast sensitivity. A principal component analysis also verified the importance of visual acuity, contrast sensitivity and visual fields in rating visibility of simulations of building elements. The required contrast for door handles, light switches and stair nosings to attain the same ratings of visibility were very similar but less contrast was required for door frames. CONCLUSIONS: 30% Michelson contrast for building elements renders building elements only poorly visible for those with severe vision impairments. 65% luminance contrast is necessary for all elements to be "easily visible". Some increase (not a decrease) on the present 30% requirement and encouragement to exceed this requirement would seem appropriate. The use of simulated objects facilitates a systematic examination of the effect of contrast, but the applicability of the results to real-life remains to be demonstrated.

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

High level of persister frequency in clinical staphylococcal isolates.

BACKGROUND: Staphylococcus aureus is a notorious human pathogen that causes often lethal systemic conditions that are mostly medical device associated biofilm infections. Similarly, coagulase negative staphylococci are emerging as leading pathogen for nosocomial infections owing to their ability to form biofilm on implanted medical equipment. Chronic in nature, these infections are difficult to treat. Such recalcitrance of these infections is caused mainly due to the presence of persister cells, which exhibit transient yet extreme tolerance to antibiotics. Despite tremendous clinical significance, there is lack of studies on persister cells formation among clinical bacterial isolates. Considering the importance of factors influencing persister formation, in this study, we evaluate the association of antibiotic tolerance with biofilm production, antibiotic stress, growth phase, specimen type, and dependency on staphylococcal species. Biofilm formation was detected among 375 clinical staphylococcal isolates by quantitative tissue culture plate method (TCP) and icaAD genes by genotypic method. The antibiotic susceptibility was determined by Kirby Bauer disc diffusion method while minimum inhibitory concentration values were obtained by agar dilution method. Persister cells were measured in the susceptible staphylococcal isolates in the presence of clinically relevant antibiotics. RESULTS: In the study, 161 (43%) S. aureus and 214 (57%) coagulase negative staphylococci (CNS) were isolated from different clinical samples. TCP method detected biofilm production in 84 (52.2%) S. aureus and 90 (42.1%) CNS isolates. The genotypic method detected icaAD genes in 86 (22.9%) isolates. Majority (> 90%) of both the biofilm producers and non-producers were sensitive to chloramphenicol and tetracycline but were resistant to penicillin. Interestingly, all isolates were sensitive to vancomycin irrespective of biofilm production. While high persister frequency was observed among all staphylococci isolates in the stationary growth phase, the persister frequency in exponential growth phase was statistically high among isolates possessing icaAD genes compared to icaAD negative isolates. CONCLUSION: The research findings provide strong evidence that the clinical staphylococcal isolates exhibit extreme antibiotic tolerance suggesting their causal link with treatment failures. Understanding the factors influencing the formation and maintenance of persister cells are of utmost important aspect to design therapeutics and control recalcitrant bacterial infections.

Recycling Waste Paper for Further Implementation: XRD, FTIR, SEM, and EDS Studies.

Recycling technology contributes to sustainability and has received considerable interest in fulfilling consumable products' social demands, including papers. Recycled fibers are the primary source of the papermaking industry. Papers, valuable daily used materials, can be further recycled for further implementation. Here, we report a simple method for recycling waste papers for further use. Our method includes re-pulping, deinking, bleaching, and papermaking. The sample and the recycled papers were characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). XRD data shows the presence of cellulose and filler minerals in the sample and the recycled papers. FTIR analysis confirmed the presence of hydroxyl, carbonyl, and methyl functional groups in the recycled papers suggesting that the deinking and bleaching did not cause any structural changes. The fibrous structures were also sustained after recycling, as confirmed by SEM studies demonstrating that the recycling was successful and the papers can be further used and recycled. EDS analysis further confirmed the filler minerals in the sample paper with a trace amount of lead, which decreased upon bleaching the paper. The structure and properties of the sample and the recycled papers were quite similar, inferring that waste papers can be recycled again and different products from low to higher grade papers can be fabricated.

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.

Issues in specifying contrast in building elements for people with a visual disability.

BACKGROUND: Standards writers, national and international, have used different contrast calculations to set requirements in building elements for people with visual impairments. On the other hand, they have typically set a single requirement (30%) for specifying the minimum contrast. The systems are not linearly related and 30% means something rather different in each system. OBJECTIVE: To provide a comparison of the various scales in order to illustrate the differences caused by multiple scales with a single compliance value, recommend a single scale for universal adoption and, if a new measure is problematic for implementation, to recommend the most perceptually uniform of the present methods. METHODS: We use the contrast between combinations of 205 paint colours to illustrate the relationships between the measures. We use an internationally accepted scale, with equal perceptual steps, as a "gold standard" to identify the most perceptually uniform measurement scale in the existing methods. RESULTS: We show that Michelson contrast is the most perceptually uniform of the existing measurement scales. We show the contrasts in the proposed method that equate to the various current requirements. CONCLUSIONS: We propose that CIE Metric Lightness could be used as the contrast measure. Alternatively, Michelson contrast is the most perceptually linear of the current measurement scales.

Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern.

BACKGROUND: Coagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns. METHODS: A total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes. RESULTS: Among all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates. CONCLUSION: The study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

Distribution of Macrolide-Lincosamide-Streptogramin B Antibiotics Resistance Genes in Clinical Isolates of Staphylococci.

BACKGROUND: Staphylococci are posing threat due to increasing trend of antimicrobial resistance particularly methicillin. Macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat such infections. This study was aimed to determine the prevalence of inducible clindamycin resistance and observation of erm and msr genes among Staphylococci isolated from tertiary care hospital of Nepal during July 2017 to March 2018. METHODS: Staphylococci from different clinical specimens were identified and antibiotic susceptibility profile was assessed following Kirby Bauer disc diffusion method. The double disc diffusion or D-zone test as outlined in CLSI document M100-S24 was performed to examine inducible clindamycin resistant isolates. Multiplex PCR was performed for detection of erm and msr gene in isolates using specific primers for ermA, ermB, ermC, msrA and msrB genes. RESULTS: Of the 60 Staphylococci isolates, 39 (65%) were S. aureus and 21 (35%) were coagulase negative Staphylococci (CNS) with 25 (64%) and 15 (71%) representing methicillin resistant S. aureus and CNS respectively. Constitutive and inducible MLSB phenotype was observed among 24 (40%) and 14 (23%) isolates respectively by D test. The most prevalent resistant gene was ermC (37%) followed by msrB (12%), ermB (10%) and msrA (10%). None of the isolates were found to possess ermA gene. CONCLUSIONS: The presence of constitutive and inducible MLSB as well as resistant genes among Staphylococci necessitates detection of such isolates to minimize treatment failure. The result from this study may help elucidate the predominant resistant characteristics in clinical Staphylococci isolated from tertiary care hospital of Nepal.

In Vitro Antimicrobial Activity of Some Medicinal Plants against Human Pathogenic Bacteria.

The emergence and spread of antibiotic resistance, as well as the evolution of new strains of disease causing agents, are of great concern to the global health community. Effective treatment of a disease entails the development of new pharmaceuticals or some potential source of novel drugs. Commonly used medicinal plants of our community could be an excellent source of drugs to fight off this problem. This study is focused on exploring the antimicrobial properties of the plants that are commonly being used as traditional medicines. The antimicrobial potential of four different plant extracts was screened against twelve pathogenic microorganisms and two reference bacterial strains. Methanolic extracts of Oxalis corniculata, Artemisia vulgaris, Cinnamomum tamala, and Ageratina adenophora were subjected to a test of their antimicrobial properties by agar well diffusion method. The result indicated that most of the extracts exhibited antimicrobial properties. The highest potential was observed in the extract of O. corniculata against Escherichia coli, Salmonella Typhi, MDR Salmonella Typhi, Klebsiella pneumoniae, and Citrobacter koseri with zone of inhibition (ZOI) of 17 mm, 13 mm, 16 mm, 11 mm, and 12 mm, respectively. Oxalis corniculata also showed the highest MIC against test organisms. The methanolic extract of Artemisia vulgaris, Cinnamomum tamala, and Ageratina adenophora showed efficacy against Staphylococcus aureus. Ageratina adenophora also showed antifungal activity against Rhizopus spp. The experiment confirmed the efficacy of some selected plant extracts as natural antimicrobials and suggested the possibility of employing them in drugs for the treatment of infectious diseases caused by the test organisms.

Biofilm Producing Clinical Staphylococcus aureus Isolates Augmented Prevalence of Antibiotic Resistant Cases in Tertiary Care Hospitals of Nepal.

Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. In this premise, we attempt to investigate the biofilm formation and its association with antibiotic resistance in clinical isolates from the patients visiting tertiary health care hospitals in Nepal. Bacterial cells isolated from clinical samples identified as S. aureus were examined for in-vitro biofilm production using both phenotypic and genotypic assays. The S. aureus isolates were also examined for susceptibility patterns of clinically relevant antibiotics as well as inducible clindamycin resistance using standard microbiological techniques and D-test, respectively. Among 161 S. aureus isolates, 131 (81.4%) were methicillin resistant S. aureus (MRSA) and 30 (18.6%) were methicillin sensitive S. aureus (MSSA) strains. Although a majority of MRSA strains (69.6%) showed inducible clindamycin resistance, almost all isolates (97% and 94%) were sensitive toward chloramphenicol and tetracycline, respectively. Detection of in vitro production of biofilm revealed the association of biofilm with methicillin as well as inducible clindamycin resistance among the clinical S. aureus isolates.

Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates.

OBJECTIVE: Staphylococcus genus comprising both Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely distributed in nature and can infect diversity of hosts. Indeed, staphylococci are the major pathogens causing biofilm associated infections caused by contaminated hospital indwelling devices. These infections are persistent in nature being highly refractory to various stresses including antibiotics. Implementation of efficient diagnostic techniques for the biofilm production would help minimize the disease burden. Thus, early detection of pathogenic strains producing biofilms warrant the utmost importance in diagnostic laboratories especially in resource limited settings. RESULT: Among 375 isolates collected from different clinical specimens, 214 (57%) were identified as coagulase negative staphylococci and 161 (43%) S. aureus. Detection of In-vitro biofilm formation in these isolates were carried out by three commonly used phenotypic assays and a genotypic assay. While evaluating the results, tissue-culture method with supplemented glucose and sucrose showed the best correlation with the results of genotypic assay.

Ocular morbidity among the children of squatter settlements in Kathmandu.

PURPOSE: To determine the prevalence of ocular morbidity and refractive error among the children of the squatter settlements in Kathmandu. METHODS: A cross-sectional study was carried out at five squatter settlement areas at Kathmandu, including 366 children younger than 16 years. Detailed eye examination included the visual acuity testing, cycloplegic refraction, binocular vision assessment, anterior segment examination, and posterior segment examination. Variations in age, sex, and ethnic distribution of the study population were analyzed through analysis of variance. Pearson χ test with Yate correction was used to analyze different types of ocular morbidity. Pearson correlation coefficient test was performed to correlate refractive error in the right eye and the left eye. RESULTS: Majority of children (28.9%) belonged to the age group 8 to 10 years, and most of them belonged to the Manohara settlement area (40.2%). Male-female ratio was 0.7. Uncorrected and best-corrected visual acuity of better than or equal to 6/9 in at least one eye was found in 87.9% and 99.0% children, respectively. Total ocular morbidity was observed in 33.7%. The common type of ocular morbidity was conjunctivitis (11.2%), refractive error (9.0%), and blepharitis (5.4%). Ocular morbidity was common in infants (57.9%; p = 0.043; odds ratio, 2.8) and 14- to 15-year-old children (52.8%; p = 0.002; odds ratio, 2.5). CONCLUSIONS: Ocular infection and refractive error represent the common ocular morbidity in children living in squatter settlements.