Screening of MMV pandemic response and pathogen box compounds against pan-drug-resistant Klebsiella pneumoniae to identify potent inhibitory compounds.

Background: The recent emergence of pan-drug-resistant (PDR) K. pneumoniae strains hinders the success rate of treatment procedures for patients. High mortality, extended duration of hospitalization with high costs is associated with such infections. Discovery and identification of new drugs are inevitable to combat PDR clinical pathogens. We aim to identify and evaluate new compounds in vitro against a PDR clinical K. pneumoniae isolate using compounds of Pathogen Box and Pandemic Response Box from Medicines for Malaria Venture (MMV). Methods: The PDR strain was initially screened with the 601 compounds from both Boxes at 10 â€‹µM concentration. Formation of dormant cells against the drug activity was assessed using persister assay. MIC was determined for the drugs inhibiting PDR K. pneumoniae during initial screening. Results: Five compounds were identified to inhibit the test strain. MMV1580854 (94.60 â€‹%), MMV1579788 (94.65 â€‹%), MMV1578574 (eravacycline; 93.13 â€‹%), MMV1578566 (epetraborole; 95.29 â€‹%) and MMV1578564 (96.32 â€‹%) were able to exhibit a higher percentage of growth inhibition. Persisters were found to be growing in a range from 104 to 107 â€‹CFU/ml. Minimum inhibitory concentrations (MIC) of all compounds were ≥ 2 â€‹µM except for MMV1579788, which had a MIC of ≥ 5 â€‹µM. Conclusion: Five novel compounds were identified against the highly evolved pan-drug-resistant K. pneumoniae. Among the five, epetraborole andMMV1578564 were identified as highly potent based on the persister frequency and MICs. The pan-drug resistant clinical isolate used in this study was found to be acting differently from the reference or wild type strains against the test compounds in a previous study.

Occurrence of mcr genes and alterations in mgrB gene in intrinsic colistin- resistant Enterobacterales isolated from chicken meat samples.

BACKGROUND: Tolerance of gram negative pathogens toward last resort colistin is mediated by mcr genes and alterations in chromosomal mgrB via modification of lipopolysaccharide through the PmrAB and PhoPQ component system. Proteus sp., Morganella sp., Neisseria sp., Burkholderia sp. and Providencia sp. are intrinsic resistant to colistin drug. Recent reports have shown that colistin intrinsic resistant organisms harbor and act as reservoirs for mcr genes. AIM: To evaluate the presence of mcr-1 to mcr-5 genes and alterations in mgrB gene in intrinsic colistin-resistant gram negative bacteria isolated from chicken meat samples in Coimbatore district, Tamil Nadu, India. METHODS: One hundred chicken meat samples were collected during 2019-20. Samples were enriched and plated on MacConkey agar supplemented with colistin (2 µg/ml). The bacterial isolates were then identified using biochemical tests. DNA were extracted from isolates using the thermal lysis method. mcr-1 to mcr-5 and mgrB genes was detected using conventional PCR and agarose gel electrophoresis methods. RESULT AND CONCLUSION: The presence of mcr-1 to mcr-5 genes was found to be 23 % (23/100). mcr-1 and mcr-5 genes were not detected in sample isolates. 17/23 samples positive for mcr genes were also found to be carrying alterations in mgrB gene. Phenotypic characterization of these isolates revealed that these bacteria were belonging to colistin intrinsic resistant gram negative bacteria such as Proteus sp., Providencia sp., and Morganella sp. Intrinsic resistant bacteria could act as a potential reservoir and disseminate mcr genes to the colistin sensitive non-intrinsic pathogens of clinical importance in the environment.

Evaluation of ELISA BP180 and BP230 Autoantibodies in Blister Fluid and Serum in the Diagnosis of Bullous Pemphigoid.

Bullous pemphigoid (BP) is a chronic subepidermal immunobullous disorder. Studies have demonstrated the presence of antibasement membrane zone antibodies (BP180 & BP230) in the blister fluid using enzyme-linked immunosorbent assay (ELISA). To detect and compare BP 180 and BP 230 autoantibodies in the blister fluid and serum of patients with BP by ELISA method. A total of 30 patients diagnosed as BP and not on treatment were included in the study. Blister fluid and serum were subjected to ELISA, and the results were compared. The sensitivity of ELISA BP 180 was found to be 95.8% in the blister fluid and 88.4% in the serum. The sensitivity of ELISA BP 230 in the blister fluid and serum was 20% and 16.6%, respectively. Association between ELISA antibodies done in blister and serum was analysed using Chi-square test and found to be statistically significant with P value <0.05. Blister fluid is an effective alternative to the serum in detecting BP 180 and BP 230 antibodies, especially in uncooperative and elderly patients with poor venous access.

Evaluation of the Role of BIOCHIP Mosaic Based Indirect Immunofluorescence and ELISA BP 180 and BP 230 Autoantibodies in the Diagnosis of Bullous Pemphigoid Patients.

Background: Bullous pemphigoid (BP) is characterized by tissue-bound and circulating Immunoglobulin G (IgG) autoantibodies against BP 180 and BP 230. Diagnosis of BP is a multi-step procedure. Enzyme-linked immunosorbent assay (ELISA) is a quantitative analysis of target antigens, whereas BIOCHIP mosaic-based indirect immunofluorescence (IIF) has a combination of screening and target antigen-specific substrates in a single miniature incubation field. This study is done to compare BIOCHIP mosaic based IIF and ELISA for the diagnosis of BP. Materials and Methods: A total of 42 biopsy and/or direct immunofluorescence (DIF) proven BP patients were included in the study. Serum was subjected to BIOCHIP mosaic-based IIF and ELISA. The results were then compared. Results: Using ELISA, the sensitivity of BP 180 and BP 230 was 92.3% and 54.5%, respectively. The sensitivity of BP 180 and BP 230 by BIOCHIP was 77.4% and 60%, respectively. The association between ELISA and BIOCHIP was analyzed using the Chi-square test and was found to be statistically significant with a P value ≤ 0.05. Conclusion: Our study concluded that both BIOCHIP and ELISA showed comparable sensitivity in diagnosing BP. Both are non-invasive, less time-consuming, and provide fast results. However, BIOCHIP can delineate bullous pemphigoid from other sub-epidermal bullous diseases.

Evaluation of the Role of Oral Mucosal Direct Immunofluorescence and Salivary Desmoglein 1 and 3 Enzyme-Linked Immunosorbent Assay in Patients With Oral Mucosal Pemphigus.

Background: Pemphigus vulgaris (PV) is characterized by antibodies against desmosomal adhesion proteins desmoglein (Dsg) 1 and 3 which can be detected by direct immunofluorescence (DIF) or enzyme-linked immunosorbent assay (ELISA). Oral lesions usually precede cutaneous lesions and an early diagnosis can prevent mortality and morbidity. Dsg antibodies can be detected by ELISA in saliva of patients with oral mucosal pemphigus. This study compares oral mucosal DIF with the salivary Dsg1 and 3 ELISA. Materials and Methods: A total of 26 biopsy and/or DIF-proven PV patients with oral erosions without cutaneous lesions were included in the study. Biopsy of oral mucosa was taken for DIF by standard method. Saliva sample was obtained and processed for ELISA. The results were then compared. Results: Out of 26 patients, 22 (84.6%) had a positive oral mucosal DIF and four patients (15.4%) had negative DIF. Nine patients (34%) had positive salivary Dsg3 ELISA. Seven patients (27%) had positive salivary Dsg1 ELISA. Taking oral DIF as the gold standard, the sensitivity of salivary Dsg1 ELISA was 31.8% and of salivary Dsg3 ELISA was 40.9%. Conclusion: Although DIF is the gold standard for the diagnosis of PV, salivary Dsg1 and 3 ELISA can also be used in the diagnosis of oral pemphigus.

Behavior of Pseudomonas aeruginosa strains on the nanopillar topography of dragonfly (Pantala flavescens) wing under flow conditions.

Bacterial associated infection is a threat in the medical field. Pseudomonas aeruginosa, one of the major causative agents for nosocomial infection, has developed resistance to almost all the classes of antibiotics. Recently, nanopillar-like structures were identified on the wings of insects such as cicada and dragonfly. Nanopillars both on natural surfaces and those mimicked on artificial surfaces were reported to possess bactericidal activity against a wide range of bacteria. An earlier study reported strain specific variation in the viability of P. aeruginosa on the nanopillar topography of a dragonfly wing under static condition. Here, we report the behavior of P. aeruginosa strains on a dragonfly wing under hydrodynamic conditions. The results of the study indicated that, under hydrodynamic conditions, P. aeruginosa PAO1 was attached in higher numbers to the wing surface than P. aeruginosa ATCC 9027 but killed in lower numbers. The plausible reason was identified to be the masking of nanopillars by the secreted extracellular polysaccharide. The shear rate of 1.0 s-1 showed a relatively higher bactericidal effect among the three tested shear rates.

Characteristics, outcome and risk factors for mortality of paediatric patients with ICU-acquired candidemia in India: A multicentre prospective study.

BACKGROUND: The epidemiology, clinical profile and outcome of paediatric candidemia vary considerably by age, healthcare settings and prevalent Candida species. Despite these differences, few comprehensive studies are undertaken. This nationwide study addresses this knowledge gap. METHODS: 487 children who contracted ICU-acquired candidemia at 23 Indian tertiary care centres were assessed for 398 variables spanning demography, clinical characteristics, microbiology, treatment and outcome. RESULTS: Both neonates (5.0 days; range = 3.0-9.5) and non-neonatal children (7.0 days; range = 3.0-13.0) developed candidemia early after ICU admission. Majority of neonates were premature (63.7%) with low birthweight (57.1%). Perinatal asphyxia (7.3%), pneumonia (8.2%), congenital heart disease (8.4%) and invasive procedures were common comorbidities, and antibiotic use (94.1%) was widespread. C tropicalis (24.7%) and C albicans (20.7%) dominated both age groups. Antifungal treatment (66.5%) and removal of central catheters (44.8%) lagged behind. Overall resistance was low; however, emergence of resistant C krusei and C auris needs attention. The 30-day crude mortality was 27.8% (neonates) and 29.4% (non-neonates). Logistic regression identified admission to public sector ICUs (OR = 5.64), mechanical ventilation (OR = 2.82), corticosteroid therapy (OR = 8.89) and antifungal therapy (OR = 0.22) as independent predictors of 30-day crude mortality in neonates. Similarly, admission to public sector ICUs (OR = 3.62), mechanical ventilation (OR = 3.13), exposure to carbapenems (OR = 2.18) and azole antifungal therapy (OR = 0.48) were independent predictors for non-neonates. CONCLUSIONS: Our findings reveal a distinct epidemiology, including early infection with a different spectrum of Candida species, calling for appropriate intervention strategies to reduce candidemia morbidity and mortality. Independent factors identified in our regression models can help tackle these challenges.

Invitro Activities of Polymyxins and Rifampicin against Carbapenem Resistant Acinetobacter baumannii at a Tertiary Care Hospital from South India.

INTRODUCTION: Acinetobacter baumannii (A.baumannii) is rapidly emerging as a potent organism causing a multitude of nosocomial infections. The organism also carries various resistance mechanisms to antibiotics, making treatment more difficult. Very few choices are left, as A.baumannii strains have begun to develop resistance against cephalosporins, aminoglycosides and even carbapenems. AIM: To examine the sensitivity pattern of three older antibiotics namely colistin, polymyxin B and rifampicin against carbapenem resistant A.baumannii by disk diffusion method and the sensitivity of colistin alone by Minimum Inhibitory Concentration (MIC) determination by VITEK automated system. MATERIALS AND METHODS: Hundred clinical isolates of carbapenem resistant A. baumannii were tested for sensitivity to colistin, polymyxin B and rifampicin by Kirby-Bauer disk diffusion method. They were also tested for sensitivity to colistin by VITEK 2C (biomérieux) automated microbial identification system. The zone diameters and Minimum Inhibitory Concentration values for the above two methods, respectively were observed and analysed. All the Antibiotic Susceptibility Tests were done according to the CLSI guidelines. RESULTS: By Kirby-Bauer disk diffusion method, 78% of the carbapenem resistant strains were found to be sensitive, 12% intermediate sensitive and 10% resistant to colistin. All the isolates were sensitive to polymyxin B and 80% were resistant to rifampicin. By the VITEK automated system, 99% of the isolates were sensitive to colistin (more in number than by disk diffusion method). CONCLUSION: Polymyxins (colistin - polymyxin E and polymyxin B) are the next choice for multidrug resistant serious nosocomial infections like those of A. baumannii, till newer antibiotics are discovered to treat such infections. Rifampicin resistance was found to be very high and hence, is not advised for monotherapy.