Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla NDM-1 Klebsiella pneumonia and bla NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

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BackgroundCryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported.ResultsWe performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing.ConclusionsProteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans.

The frequency of Candida infections is currently rising, and thus adversely impacting global health. The situation is exacerbated by azole resistance developed by fungal pathogens. Candida tropicalis is an opportunistic pathogen that causes candidiasis, for example, in immune-compromised individuals, cancer patients, and those who undergo organ transplantation. It is a member of the non-albicans group of Candida that are known to be azole-resistant, and is frequently seen in individuals being treated for cancers, HIV-infection, and those who underwent bone marrow transplantation. Although the genome of C. tropicalis was sequenced in 2009, the genome annotation has not been supported by experimental validation. In the present study, we have carried out proteomics profiling of C. tropicalis using high-resolution Fourier transform mass spectrometry. We identified 2743 proteins, thus mapping nearly 44% of the computationally predicted protein-coding genes with peptide level evidence. In addition to identifying 2591 proteins in the cell lysate of this yeast, we also analyzed the proteome of the conditioned media of C. tropicalis culture and identified several unique secreted proteins among a total of 780 proteins. By subjecting the mass spectrometry data derived from cell lysate and conditioned media to proteogenomic analysis, we identified 86 novel genes, 12 novel exons, and corrected 49 computationally-predicted gene models. To our knowledge, this is the first high-throughput proteomics study of C. tropicalis validating predicted protein coding genes and refining the current genome annotation. The findings may prove useful in future global health efforts to fight against Candida infections.

Combating antibiotic resistance has found great interest in the current clinical scenario. Pseudomonas aeruginosa , an opportunistic multidrug-resistant pathogen, is well known for its deadly role in hospital-acquired infections. Infections by P. aeruginosa are among the toughest to treat because of its intrinsic and acquired resistance to antibiotics. In this study, we project gallium–curcumin nanoparticle (GaCurNP) conjugates as a prospective candidate to fight against P. aeruginosa . The synthesized GaCurNPs were spherical with an average size ranging from 25–35 nm. Analysis by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy deduced the nature of interaction between gallium and curcumin. Conjugate formation with gallium was found to improve the stability of curcumin at the physiological pH. When tested after 24 h of contact, at the physiological pH and 37 °C, the degradation of curcumin bound in the GaCurNPs was 26%, while that of native curcumin was 95%. The minimum inhibitory concentration (MIC) of GaCurNPs was found to be 82.75 μg/mL for P. aeruginosa ( ATCC 27853). GaCurNPs also showed excellent biofilm inhibition at 4MIC concentration. Raman spectroscopic analysis showed that GaCurNPs are capable of disrupting the cells of P. aeruginosa within 3 h of contact. Live/dead imaging also confirmed the compromised membrane integrity in cells treated with GaCurNPs. Scanning electron microscopy analysis showed membrane lysis and cell structure damage. The AlamarBlue assay showed that when L929 cell lines were treated with GaCurNPs with concentrations as high as 350 μg/mL, the cell viability elicited by the nanoparticles was 70.89%, indicating its noncytotoxic nature. In short, GaCurNPs appear to be a promising antibacterial agent capable of fighting a clinically significant pathogen, P. aeruginosa .

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Background: Recent changes in the diagnostic criteria and the introduction of newer technologies like prosthetic valve replacement require the need to identify the changing epidemiology of prosthetic valve endocarditis (PVE). Materials and methods: This is a retrospective, cross-sectional, observational study. Patients diagnosed with Candida parapsilosis definite and possible PVE as per modified Duke’s criteria for a period of 11 years from January 2010 to December 2020 were included for the analysis. Results: Twelve of the 47 PVE cases (25.5%) were caused by C. parapsilosis . The median age of the patients was 52 years. Males were predominantly affected (58%). Based on the modified Duke’s criteria, eight (67%) were definite infective endocarditis (IE) cases. The single valve was affected in 11 cases (92%) with the mitral valve being the commonest ( n = 8, 67%). The type of valve commonly involved was mechanical [ n = 10, 83%]. The mean size of the vegetation was 13.15 mm. Most cases ( n = 7, 58%) were late-onset PVE. The mean C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and procalcitonin (PCT) levels for C. parapsilosis PVE were 70.2 mg/L, 51.08 mm/hour, and 0.3 ng/mL, respectively. The rates of complications and in-hospital mortality were 75% each. The most common observed complication was embolic events ( n = 8, 67%). Statistical significance ( p ≤ 0.05) was observed for mean vegetation size, overall complications, embolic events, and mortality for C. parapsilosis PVE when compared with bacterial PVE. Conclusion: C. parapsilosis was the commonest etiological agent causing PVE. Predominant mitral valve involvement, higher rates of late-onset presentation, complications, and mortality were key differential characteristics observed. Highlights: The manuscript throws light on the changing epidemiology, clinical, and microbiological profile of PVE due to Candida sp., which are scarcely studied and reported in low- and middle-income countries like India. How to cite this article: Ponnambath DK, Gopalakrishnan A, Pillai VV, Kaviyil JE, Raja K. Clinical Profile of Prosthetic Valve Endocarditis due to Candida parapsilosis : An 11-year Retrospective Observational Study from a Quaternary Cardiac Referral Institute in India. Indian J Crit Care Med 2021;25(8):860–865.