Background Diabetes is an important risk factor for developing tuberculosis. This association leads to exacerbation of tuberculosis symptoms and delayed treatment of both the diseases. Molecular mechanism and biomarkers/drug targets related to copathogenesis of tuberculosis and diabetes are still poorly understood. In this study, proteomics based 2D-MALDI/MS approach was employed to identify host signature proteins which are altered during copathogenesis of tuberculosis and diabetes. Methods Comparative proteome of human peripheral blood mononuclear cells (PBMCs) from healthy controls, tuberculosis and diabetes patients in comparison to comorbid diabetes and tuberculosis patients was analyzed. Gel based proteomics approach followed by in gel trypsin digestion and peptide identification by mass spectrometry was used for signature protein identification. Results Total of 18 protein spots with differential expression in tuberculosis and diabetes copathogenesis (TBDM) patients in comparison to other groups were identified. These proteins belonged to four functional categories i.e. structural, cell cycle/growth regulation, signaling and intermediary metabolism. These include Vimentin, tubulin beta chain protein, Actin related protein 2/3 complex subunit 2, coffilin 1 (Structural), PDZ LIM domain protein, Rho-GDP dissociation inhibitor, Ras related protein Rab (signaling), superoxide dismutase, dCTPpyrophosphatase 1, Transcription initiation factor TFIID subunit 12, three isoforms of Peptidylprolylcis-trans isomerase A, SH3 domain containing protein (metabolism), three isoforms of Protein S100A9 and S100A8 (cell cycle progression/growth regulation). Conclusion Proteins identified to be differentially expressed in TBDM patient can act as potent biomarkers and as predictors for copathogenesis of tuberculosis and diabetes.
Background and aim Multidrug resistant Klebsiella pneumoniae is associated with nosocomial infections in both outbreak and non-outbreak situations. The study intends to evaluate the potential of enterobacterial repetitive intergenic consensus- polymerase chain reaction (ERIC-PCR), a genomic based typing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) proteomic-based typing techniques for clonal relatedness among multidrug resistant Klebsiella pneumoniae isolates. Methodology Multidrug resistant clinical isolates of Klebsiella pneumoniae (n = 137) were collected from March 2019 to February 2020. Identification and protein-based phylogenetic analysis were performed by MALDI-TOF MS. Genomic typing was done by ERIC-PCR and analyzed by an online data analysis service (PyElph). Dice method with unweighted pair group method with arithmetic mean (UPGMA) program was used to compare the ERIC profiles. The samples were also evaluated by PCR for the presence of genes encoding carbapenemases, extended spectrum beta lactamases (ESBLs) and mobile colistin resistance-1 (mcr1). Result and conclusion The study presents ERIC-PCR as more robust and better discriminatory typing tool in comparison to MALDI-TOF for clonal relatedness in multidrug resistant K. pneumoniae clinical isolates. Isolates were typed into 40 ERIC types, and six groups by MALDI-TOF-MS. PCR-based analysis revealed that all the strains harbored two or more ESBL and carbapenemase genes. None of the isolates revealed the presence of the plasmid mediated mcr-1 gene for colistin resistance.
18Background: Diabetes is an important risk factor for developing tuberculosis. This association 19 leads to exacerbation of tuberculosis symptoms and delayed treatment of both the diseases. 20 Molecular mechanism and biomarkers/drug targets related to copathogenesis of tuberculosis and 2 21 diabetes, however, still remains to be poorly understood. In this study, proteomics based 2D-22 MALDI/MS approach was employed to identify host signature proteins which are altered during 23 copathogenesis of tuberculosis and diabetes. 24 Methods: Comparative proteome of human peripheral blood mononuclear cells (PBMCs) from 25 healthy controls, tuberculosis and diabetes patients in comparison to comorbid diabetes and 26 tuberculosis patients was analyzed. Gel based proteomics approach followed by in gel trypsin 27 digestion and peptide identification by mass spectrometry was used for signature protein 28 identification. 29 Results: Total of 18 protein spots with differential expression in TBDM patients in comparison 30 to other groups were identified. These include Vimentin, tubulin beta chain protein, superoxide 31 dismutase, Actin related protein 2/3 complex subunit 2, PDZ LIM domain protein, Rho-GDP 32 dissociation inhibitor, Ras related protein Rab, dCTPpyrophosphatase 1, Transcription initiation 33 factor TFIID subunit 12, coffilin 1, three isoforms of Peptidylprolylcis-trans isomerase A, three 34 isoforms of Protein S100A9, Protein S100A8 and SH3 domain containing protein. These 35 proteins belonged to four functional categories i.e. structural, cell cycle/growth regulation, 36 signaling and intermediary metabolism.40 42 10million fresh TB cases has been reported worldwide (1). Despite extensive research on the 3 43 biology of M. tuberculosis, exact mechanism of infection and immune evasion still remains 44 elusive. Copathogenesis with HIV and diabetes further complicates the tuberculosis control 45 measures. Although HIV infection is the topmost risk factor for development of active 46 tuberculosis but population attributable risk of diabetes is more than that of HIV infection (2, 3) 47 as diabetes is known to triple the risk of developing active tuberculosis (4). As diabetes is 48 associated with various immunological dysfunctions, diabetic patients fall prey to other co-49 infections like tuberculosis, melioidosis and other conventional hyperglycemia related 50 complications like various cardiovascular disorders, retinopathy, nephropathy and many more. 51 Diabetes mellitus affected 425 million individuals worldwide in 2017 and is predicted to reach 52 629 million by 2045, the time at which 80% of diabetics will be residents of economically 53 challenged countries where active tuberculosis (TB) prevails (5, 6). Countries with highest 54 burden of diabetes are also in the list of WHO's tuberculosis high burden countries and India and 55China for example, have first two positions for the copathogenesis of TB and diabetes (7, 8). The 56 epidemiological data for association between TB and DM is increasing significantly...
Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis , it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.
Introduction: β-lactam antibiotics, mainly cephalosporins, and carbapenems, have been the mainstay of treatment for infections caused by Enterobacterales. However, their role in treating clinical infections has become limited because of the increase in resistance. There is a need to have cost-effective and rapid methods for antimicrobial susceptibility testing methods for newer antibiotics like ceftazidime-avibactam against carbapenem-resistant Enterobacterales (CRE), which can be applied in routine clinical microbiology laboratories. With this aim, the present study was conducted to compare the disk diffusion and gradient diffusion, i.e., the E-test method with the reference broth microdilution (BMD) method for in-vitro testing of ceftazidime-avibactam against CRE. Material and Methods: A total of 111 CRE isolates from various clinical samples were included. Conventional PCR (Polymerase Chain Reaction) was done for the detection of genes encoding carbapenemases and to see their expression, modified carbapenem inactivation method (mCIM) along with EDTA (Ethylenediaminetetraacetic acid) carbapenem inactivation method (eCIM) was done. Results: 42.3% (47/111) isolates were resistant to ceftazidime-avibactam by the standard broth microdilution method; however, 45.9% (51/111) were resistant by both disk diffusion and E-test. In 5.4% of isolates (similar in both methods), microbroth dilution method results did not match with E-strip and disk diffusion. Very major errors (VME) by both disk diffusion and E-test were found in 2.1% (1/47), and major errors (ME) were found in 7.8% (5/64) isolates (similar isolates in both methods). The overall categorical agreement (CA) rate was 94.6% for both E-test and disk diffusion, and the essential agreement (EA) rate was 90.1% (100/111) for E-test. 98% (109/111) of CRE harbored carbapenemase genes either singly (30.3%) or in combination with others (69.7%). Conclusion: In conclusion, for CRE, E-test and the disk diffusion method for ceftazidime-avibactam depicted an acceptable performance as an alternative to the reference broth microdilution method.
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