Identification, prioritization, and evaluation of RlpA protein as a target against multidrug-resistant Pseudomonas aeruginosa.

According to the World Health Organization, infectious diseases, particularly those caused by multidrug-resistant bacteria (MDR), are projected to claim the lives of 15 million people by 2050. Septicemia carries a higher morbidity and mortality rate than infections caused by susceptible Pseudomonas aeruginosa, and MDR-mediated ocular infections can lead to impaired vision and blindness. To identify and develop a potential drug against MDR P. aeruginosa, we employed in silico reverse genetics-based target mining, drug prioritization, and evaluation. Rare Lipoprotein A (RlpA) was selected as the target protein, and its crystal structure was geometrically optimized. Molecular docking and virtual screening analyses revealed that RlpA exhibits strong binding affinity with 11 compounds. Among these, 3-chlorophthalic acid was evaluated, and subsequent in vitro assays demonstrated significant anti-Pseudomonas activity with negligible cytotoxicity. The compound was further evaluated against both drug-susceptible and MDR P. aeruginosa strains in vitro, with cytotoxicity assessed using an MTT assay. The study demonstrated that 3-chlorophthalic acid exhibits potent anti-Pseudomonas activity with minimal toxicity to host cells. Consequently, this compound emerges as a promising candidate against MDR P. aeruginosa, warranting further investigation.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Author Correction: Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens.

The original publication of this article [1] was missing the below author that made contributions to the research and the published article.

First Report on Detection of Dengue Virus in the Donor Cornea.

BACKGROUND: Dengue fever is the most prevalent form of flavivirus infection in humans. We have investigated whether corneoscleral tissue of the donor affected by dengue virus (DENV) harbors the virus. PURPOSE: To identify the risk for viral transmission through corneal transplants in areas where DENV circulates. METHODS: Excised corneoscleral tissue from a cadaver with a history of viral hemorrhagic fever was analyzed using reverse transcriptase-polymerase chain reaction for the presence of DENV and chikungunya virus (CHIV). RESULTS: DENV was detected in RNA extracted from the donor corneoscleral rim. Further genotyping of the viral isolate from the virus-infected cell harvest revealed DENV type 3 as the causative agent. CHIV was not detected. CONCLUSIONS: The data presented in this study recommend the implementation of polymerase chain reaction for detection of DENV and CHIV to analyze excised corneoscleral tissue of a donor with viral hemorrhagic fever.

Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens.

Human adenoviruses (HAdVs) are uniquely important "model organisms" as they have been used to elucidate fundamental biological processes, are recognized as complex pathogens, and are used as remedies for human health. As pathogens, HAdVs may effect asymptomatic or mild and severe symptomatic disease upon their infection of respiratory, ocular, gastrointestinal, and genitourinary systems. High-resolution genomic data have enhanced the understanding of HAdV epidemiology, with recombination recognized as an important and major pathway in the molecular evolution and genesis of emergent HAdV pathogens. To support this view and to actualize an algorithm for identifying, characterizing, and typing novel HAdVs, we determined the DNA sequence of 95 isolates from archives containing historically important pathogens and collections housing currently circulating strains to be sequenced. Of the 85 samples that were completely sequenced, 18 novel recombinants within species HAdV-B and D were identified. Two HAdV-D genomes were found to contain novel penton base and fiber genes with significant divergence from known molecular types. In this data set, we found additional isolates of HAdV-D53 and HAdV-D58, two novel genotypes recognized recently using genomics. This supports the thesis that novel HAdV genotypes are not limited to "one-time" appearances of the prototype but are of importance in HAdV epidemiology. These data underscore the significance of lateral genomic transfer in HAdV evolution and reinforce the potential public health impact of novel genotypes of HAdVs emerging in the population.

Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes.

Pseudomonas aeruginosa (P. aeruginosa) is a menacing opportunistic, nosocomial pathogen; become a growing concern as conventional antimicrobial therapy is now futile against it. Multi-drug resistant P. aeruginosa (MDRPA) has distinctive resistance mechanisms such as production of ß-lactamases, repression of porin genes and over-expression of efflux pumps. The focus of this study is to standardize and application of multiplex PCR (mPCR) to detect the presence of betalactamase genes encoding blaTem, blaOXA, blaCTX-M-15, blaVim, blaGes, blaVeb, blaDIM, AmpC and Efflux pump genes encoding Mex A,B-oprM, Mex C,D-oprJ, Mex X,Y-oprN, oprD, nfxB, MexR. A total of 200 clinical isolates of P. aeruginosa were tested for the presence of the above mentioned genes genotypically through mPCR and characterized by phenotypic methods for ESBL and MBL production. Out of 200 isolates, 163 (81.5%) nfxB regulator gene, 102 (51%) MexA, 96 (48%) MexC, 93 (46.5%) MexB, 86 (43%) MexD, 81 (40.5%) OprM, 74 (37%) OprJ, 72 (36%) OprD and MexR, 53 (26.5%) Mex X and OprN, 49 (24.5%) MexY gene. Betalactamase genes 145 (72.5%) blaTem, 67 (33.5%) blaOXA, 35 (17.5%) blaVim, 25(12.50%), 23 (11.50%) blaVeb, 21 (11.5%) blaGes, 14 (7%) Ctx-m and 10 (5%) AmpC and 5 (2.5%) blaDim-1 gene were tested positive by mPCR. Phenotypically 38 (19%) and 29 (14.5%) out of 200 tested positive for ESBL and MBL production. Application of this mPCR on clinical specimens is fast, accurate, specific and low-cost reliable tool for the screening, where culture negative Eubacterial PCR positive cases for an early molecular detection of drug resistance mechanism assisting the clinician to treat the disease with appropriate antibiotic selection.

Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study.

PURPOSE: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon's capsule fibroblast (HTFs) in an in vitro model. PATIENTS AND METHODS: Tenon's capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. RESULTS: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5-2.0%), the viability of cells decreased from 1.0% lignocaine. CONCLUSION: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

Frosted Branch Angiitis in a Patient with Typhoid Fever.

Frosted branch angiitis (FBA), a rare form of retinal vasculitis presenting as bilateral perivascular sheathing, resembling the appearance of frosted tree branches in winter, was first reported by Ito et al.1 in 1976, in a young immunocompetent boy. FBA predominantly affects healthy young patients, the youngest reported in an 11-month-old infant2 and oldest in a 42-year-old patient.3 Classical symptoms include sudden onset of blurred vision with floaters and photopsiae. Fundus examination shows widespread perivascular translucent sheathing affecting both arterioles and venules, more commonly latter. Fluorescein angiography shows late staining of vessels with no obstruction of blood flow. Electroretinogram shows reduced amplitude and visual fields show generalized constriction. Medline search did not show any case of frosted branch angiitis in a patient with typhoid fever.

Virulence genome analysis of Pseudomonas aeruginosa VRFPA10 recovered from patient with scleritis.

Infectious keratitis is a major cause of blindness, next to cataract and majority of cases are mainly caused by gram negative bacterium Pseudomonas aeruginosa (P. aeruginosa). In this study, we investigated a P. aeruginosa VRFPA10 genome which exhibited susceptibility to commonly used drugs in vitro but the patient had poor prognosis due to its hyper virulent nature. Genomic analysis of VRFPA10 deciphered multiple virulence factors and P.aeruginosa Genomic Islands (PAGIs) VRFPA10 genome which correlated with hyper virulence nature of the organism. The genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers LFMZ01000001-LFMZ01000044.

Resistome and pathogenomics of multidrug resistant (MDR) Pseudomonas aeruginosa VRFPA03, VRFPA05 recovered from alkaline chemical keratitis and post-operative endophthalmitis patient.

Eye infections due to Pseudomonas aeruginosa is an important cause of ocular morbidity. We presents the whole genomic comparative analysis of two P. aeruginosa VRFPA03 and VRFPA05 isolated from alkaline chemical injury mediated keratitis and post-cataract surgery endophthalmitis patients, respectively. The blaDIM-1 gene in VRFPA03 and the blaGes-9 gene in VRFPA05 were identified and reported for the first time from an ocular isolate. The current study revealed novel integrons In1107 and In1108, comprised of multidrug-resistant genes. Ocular virulence factors mainly mediated by exoenzymes T, Y, and U and exotoxin A, elastase B, and phenazine-specific methyltransferase. Genomic analysis uncovered multiple known and unknown factors involved in P. aeruginosa mediated ocular infection, which may lead to drug discovery and diagnostic markers to improve human vision care.

Stepwise Protocol for Cytospin-enhanced Smearing for Scraped Corneal Epithelial Cells.

Proteins and antigens present on the cell surface are usually determined by immunofluorescence staining. Uniform distribution of cells is required to appreciate the presence of surface proteins. Improper smearing or crushing of the corneal epithelial cells can potentially destroy the cellular integrity. Thus a simplified, systemic method was designed to smear the cells scraped from the cornea. The procedure includes trypsinisation for dissociation of corneal epithelial cells and cytospinning for concentrating the cells in a smear. The standardized protocol was found to be efficient in maintaining the integrity of the corneal epithelial cells and also the distribution of the cells in the smear.

A study on the characterization of Propionibacterium acnes isolated from ocular clinical specimens.

BACKGROUND & OBJECTIVES: There are only a few reports available on characterization of Propionibacterium acnes isolated from various ocular clinical specimens. We undertook this study to evaluate the role of P. acnes in ocular infections and biofilm production, and also do the phylogenetic analysis of the bacilli. METHODS: One hundred isolates of P. acnes collected prospectively from ocular clinical specimens at a tertiary care eye hospital between January 2010 and December 2011, were studied for their association with various ocular disease conditions. The isolates were also subjected to genotyping and phylogenetic analysis, and were also tested for their ability to produce biofilms. RESULTS: Among preoperative conjunctival swabs, P. acnes was a probably significant pathogen in one case; a possibly significant pathogen in two cases. In other clinical conditions, 13 per cent isolates were probably significant pathogens and 38 per cent as possibly significant pathogens. The analysis of 16S rRNA gene revealed four different phylogenies whereas analysis of recA gene showed two phylogenies confirming that recA gene was more reliable than 16S rRNA with less sequence variation. Results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) had 100 per cent concordance with phylogenetic results. No association was seen between P. acnes subtypes and biofilm production. INTERPRETATION & CONCLUSIONS: RecA gene phylogenetic studies revealed two different phylogenies. RFLP technique was found to be cost-effective with high sensitivity and specificity in phylogenetic analysis. No association between P. acnes subtypes and pathogenetic ability was observed. Biofilm producing isolates showed increased antibiotic resistance compared with non-biofilm producing isolates.

Genotypic Detection of Epstein Barr Virus in Pediatric Transplant Recipients From India.

OBJECTIVE: To determine the rate of occurrence and genotypes of Epstein-Barr Virus (EBV) among pediatric renal and liver transplants recipients. DESIGN: Observational study. SETTING: Vision Research Foundation referral center and Institute of Liver Disease and Transplantation, Chennai, India. PARTICIPANTS: 70 pediatric solid organ transplant recipients and 60 voluntary healthy donors. METHODS: Polymerase chain reaction (PCR) for detection and genotyping of EBV were carried out using genes targeting Viral capsid antigen, Nuclear antigen 1, 2 and 3, followed by real time PCR for viral load determination and further confirmed by phylogenetic analysis. RESULTS: EBV was detected in 35 (51.4%) samples (32 liver and 4 renal transplants) with high viral load. Type A was detected in 33 samples, Type B in 2 liver transplant patients, and co-infection in one liver transplant patient who developed Post-transplant Lymphoproliferative Disorder (PTLD). Real time PCR results correlated with conventional PCR. The mean viral load for patients who did not develop PTLD was 50,424 copies/mL. Overall EBV load in patient with PTLD ranged from 1,40,392 copies/mL prior to PTLD diagnosis to 62,124 copies /mL post treatment. CONCLUSION: EBV infection is the high risk factor for PTLD after liver transplantation. PCR targeting of EBV can be applied to diagnose EBV infections and monitor treatment for EBV in pediatric solid organ transplant recipients.

A hospital based pilot study on Epstein-Barr virus in suspected infectious mononucleosis pediatric patients in India.

INTRODUCTION: Infectious mononucleosis (IM) caused by the Epstein-Barr virus (EBV) is commonly diagnosed by detection of antibodies in the patient's sera. Differentiation of acute from chronic and differential diagnosis of EBV-induced IM from IM-like syndrome caused by human cytomegalovirus (CMV) is important. The objective of this study was to standardize and use polymerase chain reaction (PCR) for diagnosis of EBV and evaluate it against enzyme-linked immunosorbent assay (ELISA). METHODOLOGY: ELISA for detection of IgM and IgG antibodies to viral capsid antigen (VCA) and PCR targeting the VCA and EBNA1 gene of EBV and mtrII gene of CMV were performed on180 peripheral blood samples collected from 180 patients with suspected IM. The analytical sensitivity of PCR was evaluated against that of ELISA. RESULTS: Using the standard serological profile as the reference, the EBV-VCA gene was detected in 41 (95%) of 45 samples collected from patients with early primary infections, in 41 (54%) of 75 with recent primary infections, and in7 (17%) of 39 with past infections. The result of VCA PCR was statistically significant in virus detection during early or primary stage of infection. Nineteen (49%) EBV-seropositive samples were positive for CMV by PCR. All control samples tested negative for both VCA and EBNA1by PCR. CONCLUSIONS: VCA PCR is sensitive for the detection of EBV DNA in the early or primary stage of infection and can be considered a reliable method to rule out the cross-reactivity and differential diagnosis of EBV-induced IM from IM-like syndrome.

Draft genome sequence of blaVeb-1, blaoxa-10 producing multi-drug resistant (MDR) Pseudomonas aeruginosa strain VRFPA09 recovered from bloodstream infection.

Pseudomonas aeruginosa (P. aeruginosa) bacteremia causes significant mortality rate due to emergence of multidrug resistant (MDR) nosocomial infections. We report the draft genome sequence of P. aeruginosa strain VRFPA09, a human bloodstream isolate, phenotypically proven as MDR strain. Whole genome sequencing on VRFPA09, deciphered betalactamase encoding blav(eb-1) and bla(OXA-10) genes and multiple drug resistance, virulence factor encoding genes.

Draft genome sequence of blaVeb-1, blaoxa-10producing multi-drug resistant (MDR) Pseudomonas aeruginosastrain VRFPA09 recovered from bloodstream infection

Pseudomonas aeruginosa (P. aeruginosa) bacteremia causes significant mortality rate due to emergence of multidrug resistant (MDR) nosocomial infections. We report the draft genome sequence of P. aeruginosa strain VRFPA09, a human bloodstream isolate, phenotypically proven as MDR strain. Whole genome sequencing on VRFPA09, deciphered betalactamase encoding bla_veb-1 and bla_OXA-10genes and multiple drug resistance, virulence factor encoding genes.

Hepatitis C Virus NS3 Mediated Microglial Inflammation via TLR2/TLR6 MyD88/NF-κB Pathway and Toll Like Receptor Ligand Treatment Furnished Immune Tolerance.

BACKGROUND: Recent evidence suggests the neurotrophic potential of hepatitis C virus (HCV). HCV NS3 protein is one of the potent antigens of this virus mediating inflammatory response in different cell types. Microglia being the immune surveillance cells in the central nervous system (CNS), the inflammatory potential of NS3 on microglia was studied. Role of toll like receptor (TLR) ligands Pam2CSK3 and Pam3CSK4 in controlling the NS3 mediated microglial inflammation was studied using microglial cell line CHME3. METHODS: IL (Interleukin)-8, IL-6, TNF-α (Tumor nicrosis factor alpha) and IL-1ß gene expressions were measured by semi quantitative RT-PCR (reverse transcription-PCR). ELISA was performed to detect IL-8, IL-6, TNF-α, IL-1ß and IL-10 secretion. FACS (Flourescent activated cell sorting) was performed to quantify TLR1, TLR2, TLR6, MyD88 (Myeloid differntiation factor 88), IkB-α (I kappaB alpha) and pNF-κB (phosphorylated nuclear factor kappaB) expression. Immunofluorescence staining was performed for MyD88, TLR6 and NF-κB (Nuclear factor kappaB). Student's t-test or One way analysis of variance with Bonferoni post hoc test was performed and p < 0.05 was considered significant. RESULTS: Microglia responded to NS3 by secreting IL-8, IL-6, TNF-α and IL-1ß via TLR2 or TLR6 mediated MyD88/NF-κB pathway. Transcription factor NF-κB was involved in activating the cytokine gene expression and the resultant inflammatory response was controlled by NF-κB inhibitor, Ro106-9920, which is known to down regulate pro-inflammatory cytokine secretion. Activation of the microglia by TLR agonists Pam3CSK4 and Pam2CSK4 induced immune tolerance against NS3. TLR ligand treatment significantly down regulated pro-inflammatory cytokine secretion in the microglia. IL-10 secretion was suggested as the possible mechanism by which TLR agonists induced immune tolerance. NS3 as such was not capable of self-inducing immune tolerance in microglia. CONCLUSION: In conclusion, NS3 protein was capable of activating microglia and the inflammatory response could be controlled via blocking the transcription factor NF-κB, or by treating the microglia with TLR ligands which likely function via secreting anti-inflammatory cytokines such as IL-10. This can have therapeutic potential in controlling HCV mediated neuroinflammation.

Patients with dry eye without hepatitis C virus infection possess the viral RNA in their tears.

PURPOSE: Dry eye is one of the suggested extrahepatic complications associated with hepatitis C virus (HCV) infection. HCV RNA has been detected from the tear fluid of patients with chronic HCV. There has been no literature evidence on the presence of HCV RNA in the tear fluid of patients with dry eye without HCV infection. In this study, tear fluid of patients with dry eye with no HCV infection was screened for the presence of HCV RNA. METHODS: Tear fluid was collected from patients with dry eye (n = 36) and healthy controls (n = 20). Real-time polymerase chain reaction was performed to detect HCV RNA in the tear fluid. Anti-HCV enzyme-linked immunosorbent assay, alkaline phosphatase, and alanine aminotransferase tests were performed in the serum samples collected from 15 patients with dry eye. RESULTS: Viral RNA was detected in 58.3% of the patients. Serum samples collected from 15 patients with dry eye were negative for anti-HCV. Alkaline phosphatase levels were elevated in 12 of 15 patients. Alanine aminotransferase levels were normal in all 15 patients. The odds ratio for the presence of HCV RNA in patients with dry eye was 22.4. CONCLUSIONS: These results indicate a direct correlation between dry eye and HCV in non-HCV patients.

Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India.

PURPOSE: To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. METHODS: 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. RESULTS: EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. CONCLUSION: The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples.

Hepatitis C virus core and NS3 antigens induced conjunctival inflammation via toll-like receptor-mediated signaling.

PURPOSE: Dry eye condition is an extrahepatic manifestation associated with chronic hepatitis C virus (HCV) infection. Since conjunctival inflammation can contribute to the dry eye condition, in the present study we analyzed the conjunctival inflammatory response to HCV core and NS3 proteins. METHODS: We used primary human conjunctival fibroblasts for our study. Cytokines were measured with enzyme-linked immunosorbent assay (ELISA). Toll-like receptor (TLR) and cell adhesion molecule gene expression patterns were analyzed with semiquantitative reverse transcription (RT)-PCR. Immunofluorescence staining was performed for the MyD88, nuclear factor-kappa B (NF-kB), and inducible nitric oxide synthase (iNOS) proteins. Nitric oxide (NO) was measured with the Griess assay; terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed for apoptosis and cell viability, respectively. RESULTS: When exposed to the HCV core and NS3 proteins, the conjunctival fibroblasts secreted interleukin-8 (IL-8), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10 in a dose-dependent manner. Various TLRs were involved in the innate immune response via MyD88 signaling without NF-kB involvement. The gene expression of cell adhesion molecules such as CD44 and ICAM-1 was upregulated, and the cells secreted NO via iNOS. As the sum of these stress responses, the cells underwent apoptosis, which eventually lead to cell death. CONCLUSIONS: HCV core and NS3 proteins induced conjunctival inflammation that may form the pathogenesis of dry eye condition.