Unmasking the Hidden Invader: A case report of chest wall tuberculosis presenting as an abscess.

Chest wall tuberculosis is a very rare clinical entity that accounts for 1-5% of musculoskeletal tuberculosis (TB). Here we present a case of chest wall TB. A 16-year-old boy presented with complaints of progressive increasing swelling over the right hemithorax following a history of trauma. Radiologically it was a localized liquid collection with no connection to the thoracic cavity. Pus sample was sent to the mycobacteriology laboratory. The Gene Xpert result came as positive, and sensitive to rifampicin while the acid-fast bacilli (AFB) smear test was negative. Diagnosing a case of chest wall TB is always challenging for clinicians.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae.

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.

Comparative Analysis of Clinical and Genomic Characteristics of Hypervirulent Klebsiella pneumoniae from Hospital and Community Settings: Experience from a Tertiary Healthcare Center in India.

Hypervirulent Klebsiella pneumoniae (hvKp) is a hypermucoviscous phenotype of classical Klebsiella pneumoniae (cKp) that causes serious infections in the community. The recent emergence of multidrug-resistant hvKp isolates (producing extended-spectrum beta-lactamases and carbapenemases) along with other virulence factors in health care settings has become a clinical crisis. Here, we aimed to compare the distribution of virulence determinants and antimicrobial resistance (AMR) genes in relation to various sequence types (STs) among the clinical hvKp isolates from both settings, to reinforce our understanding of their epidemiology and pathogenic potential. A total of 120 K. pneumoniae isolates confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry were selected. hvKp was phenotypically identified by string test and genotypically confirmed by the presence of the iucA gene using PCR. Molecular characterization of hvKp isolates was done by whole-genome sequencing (WGS). Of the K. pneumoniae isolates, 11.6% (14/120) isolates were confirmed as hvKp by PCR (9.1% [11/120] string positive and 3.3% [4/120] positive by both methods); these were predominantly isolated from bloodstream infection (50%, 7/14), urinary tract infection (29%, 4/14), and respiratory tract infection (21%, 3/14). For all 14 hvKp infections, for 14.2% the source was in the community and for 85.7% the source was a health care setting. Two virulent plasmids were identified by WGS among the hvKp isolates from both settings. K64 was found to be the commonest capsular serotype (28.5%, 4/14), and ST2096 was the most common ST (28.5%, 4/14) by WGS. Two new STs were revealed: ST231 (reported to cause outbreaks) and ST43. The genome of one isolate was determined to be carrying AMR genes (blaCTX-M-15, blaNDM-1, blaNDM-5, blaOXA-181, blaOXA-232, etc.) in addition to virulence genes, highlighting the clonal spread of hvKp in both community and health care settings. IMPORTANCE To date, studies comparing the genomic characteristics of hospital- and community-acquired hvKp were very few in India. In this study, we analyzed the clinical and genomic characteristics of hvKp isolates from hospital and community settings. ST2096 was found as the most common ST along with novel STs ST231 and ST43. Our study also revealed the genome is simultaneously carrying AMR as well as virulence genes in isolates from both settings, highlighting the emergence of MDR hvKp STs integrated with virulence genes in both community and health care settings. Thus, hvKp may present a serious global threat, and essential steps are needed to prevent its further dissemination.

Natural products as geroprotectors: An autophagy perspective.

Over the past decade, significant attention has been given to repurposing Food and Drug Administration approved drugs to treat age-related diseases. In contrast, less consideration has been given to natural bioactive compounds. Consequently, there have been limited attempts to translate these compounds. Autophagy is a fundamental biological pathway linked to aging, and numerous strategies to enhance autophagy have been shown to extend lifespan. Interestingly, there are a number of natural products that are reported to modulate autophagy, and here we describe a number of them that activate autophagy through diverse molecular and cellular mechanisms. Among these, Urolithin A, Spermidine, Resveratrol, Fatty Acids and Phospholipids, Trehalose and Lithium are featured in detail. Finally, we outline possible strategies to optimise and increase the translatability of natural products, with the overall aim of delaying the ageing process and improving human healthspan.

Evolution of the RNase P RNA structural domain in Leptospira spp.

We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along with sequences determined from 8 reference strains were examined to fathom strain specific structural differences present in leptospiral RPR. Sequence variations in the RPR gene impacted on the configuration of loops, stems and bulges found in the RPR highlighting species and strain specific structural motifs. In vitro transcribed leptospiral RPR ribozymes are demonstrated to process pre-tRNA into mature tRNA in consonance with the positioning of Leptospira in the taxonomic domain of bacteria. RPR sequence datasets used to construct a phylogenetic tree exemplified the segregation of strains into their respective lineages with a (re)speciation of strain SH 9 to Leptospira borgpetersenii, strains Fiocruz LV 3954 and Fiocruz LV 4135 to Leptospira santarosai, strain CBC 613 to Leptospira kirschneri and strain HAI 1536 to Leptospira noguchii. Furthermore, it allowed characterization of an isolate P2653, presumptively characterized as either serovar Hebdomadis, Kremastos or Longnan to Leptospira weilii, serovar Longnan.

In vitro selection of external guide sequences for directing human RNase P to cleave a target mRNA.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence that is complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. Recent studies indicate that increasing the targeting activity of EGSs in directing human RNase P to cleave an mRNA in vitro can lead to better efficacies of the EGSs in inducing RNase P-mediated inhibition of the expression of the target mRNA in cultured cells. This chapter will describe the procedure for the generation of highly functional EGSs by in vitro selection. We also describe protocols for in vitro evaluation of the activity of the EGSs. These methods should provide general guidelines for using in vitro selection for generating highly active EGSs for gene-targeting applications.

Engineering of RNase P ribozyme for gene-targeting applications.

Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy.