Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-µPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-µPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-µPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-µPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Dual phage-incorporated electrospun polyvinyl alcohol-eudragit nanofiber matrix for rapid healing of diabetic wound infected by Pseudomonas aeruginosa and Staphylococcus aureus.

Diabetic wound healing remains a healthcare challenge due to co-occurring multidrug-resistant (MDR) bacterial infections and the constraints associated with sustained drug delivery. Here, we integrate two new species of phages designated as PseuPha1 and RuSa1 respectively lysing multiple clinical MDR strains of P. aeruginosa and S. aureus into a novel polyvinyl alcohol-eudragit (PVA-EU†) nanofiber matrix through electrospinning for rapid diabetic wound healing. PVA-EU† evaluated for characteristic changes that occurred due to electrospinning and subjected to elution, stability and antibacterial assays. The biocompatibility and wound healing ability of PVA-EU† were assessed through mouse fibroblast cell line NIH3T3, followed by validation through diabetic mice excision wound co-infected with P. aeruginosa and S. aureus. The electrospinning resulted in the incorporation of ~ 75% active phages at PVA-EU†, which were stable at 25 °C for 30 days and at 4 °C for 90 days. PVA-EU† showed sustained release of phages for 18 h and confirmed to be detrimental to both mono- and mixed-cultures of target pathogens. The antibacterial activity of PVA-EU† remained unaltered in the presence of high amounts of glucose, whereas alkaline pH promoted the activity. The matrix exerted no cytotoxicity on NIH3T3, but showed significant (p < 0.0001) wound healing in vitro and the process was rapid as validated through a diabetic mice model. The sustained release, quick wound closure, declined abundance of target MDR bacteria in situ and histopathological signs of recovery corroborated the therapeutic efficacy of PVA-EU†. Taken together, our data signify the potential application of PVA-EU† in the rapid treatment of diabetic wounds without the aid of antibiotics.

Diagnosis of Neglected Tropical Zoonotic Disease, Leptospirosis in a Clinical Sample Using a Photothermal Immunosensor.

Photothermal biosensing based on nanomaterials has gained increasing attention because of its universality and simplicity. Diagnostics of neglected tropical diseases (NTDs) in low-resource settings are challenging in terms of speed, accuracy, and cost-effectiveness. By exploiting the photothermal property of carbon nanotubes (CNTs), simple thermometric measurements can be used to generate quantitative biochemical readouts. Herein, a photothermal immunosensor for leptospirosis detection based on a CNT-labeled monoclonal antibody is established through the sensitive monitoring of the target biomarker LipL32 with a simple thermometer. Under optimum conditions, a linear range up to 106 pg/mL with a limit of detection (LOD) of 300 fg/mL was obtained. Overall, the proposed immunoassay exhibited good precision, selectivity, and acceptable stability. Clinical patient sample analysis with the photothermal sensor proved the differential diagnosis of leptospirosis along with other febrile illnesses. On the other hand, we have also characterized the photothermal sensor platform with surface morphological and spectral techniques to confirm the robust and successful fabrication of the immunosensor. The fabricated photothermal sensor could be used as a potential diagnostic tool for the early detection of NTDs in patients from resource-limited settings, as it does not require sample pretreatment, sophisticated equipment, or skilled labor. Moreover, the developed photothermal assay follows ASSURED criteria, very crucial for diagnosis in resource-limited settings.

Dysbiosis of the oral microbiota composition is associated with oral squamous cell carcinoma and the impact of radiotherapy: a pilot study.

Radiotherapy can potentially influence the diversity and composition of the oral microbiome. We performed a study comparing the composition of oral microbiota in patients with oral squamous cell carcinoma (OSCC) before radiotherapy (n = 6), at three months (n = 6), and six months (n = 6) post-radiotherapy, and controls (n = 6). We profiled the oral microbiome by 16S rRNA gene sequencing using Illumina MiSeq. Alpha diversity (Chao1 index) showed significant differences in species richness between healthy controls and OSCC patients (P = 0.014). Conversely, no noteworthy distinctions were observed in the Chao1 index when comparing the pre-and post-radiation periods at both three and six months. The beta diversity of the oral microbiota differed significantly between the controls and OSCC patients (P = 0.014). However, no significant differences were observed in beta diversity between pre- and post-radiation at three months, whereas a significant difference was observed at six months (P = 0.038). Linear Discriminant Analysis Effect Size (LEfSe) demonstrated lower abundance of Corynebacterium, Actinomyces, Veillonella, and Haemophilus, and higher abundance of Selenomonas and Mycoplasma in OSCC patients than in healthy controls. The oral microbiome composition varied among healthy controls, patients with OSCC, and post-radiation therapy patients with OSCC. The observed recovery in the numerical dominance of specific beneficial oral taxa and the reduction in pathogenic bacteria after radiation therapy highlights the need for further investigations into their clinical implications.

Description and host-range determination of phage PseuPha1, a new species of Pakpunavirus infecting multidrug-resistant clinical strains of Pseudomonas aeruginosa.

A new phage PseuPha1, infecting multiple multi-drug resistant strains of Pseudomonas aeruginosa with strong anti-biofilm activities, was isolated from wastewater in India. PseuPha1 showed optimal multiplicity of infection at 10-3, maintained the infectivity at wide ranges of pH (6-9) and temperature (4-37 °C), and exhibited 50 minutes latent period and a burst size of 200 when tested against P. aeruginosa PAO1. PseuPha1 shared 86.1-89.5% pairwise intergenomic similarity with Pakpunavirus species (n = 11) listed by the International Committee on Taxonomy of Viruses and established distinct phyletic lineages during phylogenetic analyses of phage proteins. While genomic data validated the taxonomic novelty and lytic attributes of PseuPha1, BOX-PCR profiling asserted the genetic heterogeneity of susceptible clinical P. aeruginosa. Our data supported the affiliation of PseuPha1 as a new Pakpunavirus species and provided the first line of evidence for its virulence and infectivity that can be harnessed in wound therapeutics.

Genomic potential for exopolysaccharide production and differential polysaccharide degradation in closely related Alteromonas sp. PRIM-21 and Alteromonas fortis 1T.

Members of the genus Alteromonas are widely distributed in diverse marine environments and are often associated with marine organisms. Their ability to produce exopolysaccharides (EPS) and depolymerize sulfated algal polysaccharides has provided industrial importance to some species. Here, we describe the draft genome of an algae-associated strain namely, Alteromonas sp. PRIM-21 isolated from the southwest coast of India to understand the EPS biosynthetic pathways as well as polysaccharide depolymerization system in comparison to the closely related strain Alteromonas fortis 1T that shares 99.8% 16S rRNA gene sequence similarity. Whole-genome shotgun sequencing of Alteromonas sp. PRIM-21 yielded 50 contigs with a total length of 4,638,422 bp having 43.86% GC content. The resultant genome shared 95.9% OrthoANI value with A. fortis 1 T, and contained 4125 predicted protein-coding genes, 71 tRNA and 10 rRNA genes. Genes involved in Wzx/Wzy-, ABC transporter- and synthase-dependent pathways for EPS production and secretion were common in both Alteromonas sp. PRIM-21 and A. fortis 1T. However, the distribution of carbohydrate-active enzymes (CAZymes) was heterogeneous. The strain PRIM-21 harbored polysaccharide lyases for the degradation of alginate, ulvan, arabinogalactan and chondroitin. This was further validated from the culture-based assays using seven different polysaccharides. The depolymerizing ability of the bacteria may be useful in deriving nutrients from the biopolymers produced in the algal host while the EPS biosynthesis may provide additional advantages for life in the stressful marine environment. The results also highlight the genetic heterogeneity in terms of polysaccharide utilization among the closely related Alteromonas strains.

Urease-negative uropathogen Kalamiella piersonii YU22 metabolizes urea by urea carboxylase and allophanate hydrolase enzyme system.

Urea is one of the major components of the human urine and its breakdown by the uropathogens occurs mainly through the activity of the enzyme urease. However, a few reports suggest the presence of an alternate enzyme system for urea breakdown namely urea carboxylase (UC) and allophanate hydrolase (AH). We have previously reported the UC and AH system in the genome of a urease-negative uropathogen Kalamiella piersonii YU22 of the novel genus Kalamiella (reclassified recently as Pantoea).To validate the UC and AH activity in the presence of urea, we investigated the growth and urea utilization patterns of this bacterium. Growth kinetics, variations in media pH, NH4-N generation and UC and AH gene expressions were probed using urea-containing media. YU22 was able to grow in M9 media containing urea and increase the pH of the media due to the urea breakdown. Further, significantly higher concentrations of extracellular NH4-N (p < 0.001) was also detected in the cultures along with over-expression of UC and AH genes. The bacterium formed biofilm, and displayed swimming and swarming motilities in presence of urea. Additional glucose supply to urea boosted the colonization but ameliorated the media alkalization and ammonification through suppression of gene expressions encoding UC and AH. These results show that the urease-negative strain YU22 can utilize the UC and AH system for urea metabolism. We propose to further investigate the UC and AH system in other urease-negative uropathogens and its implications for pathogenicity and urinary tract colonization.

Potential synergistic activity of quercetin with antibiotics against multidrug-resistant clinical strains of Pseudomonas aeruginosa.

Development of drug resistance in opportunistic pathogens is one of the major healthcare challenges associated with infection management. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. However, selection of the drugs should offer safety and synergistic interaction against most of the strains. Here, the efficacy of antibiotics in combination with quercetin, a natural flavonoid capable of targeting quorum sensing was tested against biofilm-forming Pseudomonas aeruginosa strains previously isolated from catheter associated urinary tract infection. Based on the antibiotic susceptibility pattern, synergistic effect of quercetin with selected antibiotics (levofloxacin, ceftriaxone, gentamycin, tobramycin and amikacin) was tested at the fractional concentrations of MIC by the checkerboard method and the fractional inhibitory concentration index (FICi) was calculated to estimate the synergistic effect. Effect of the synergistic combinations were further tested using time-kill assay, and against biofilm formation and biofilm cell viability. Cytotoxicity assays were performed using Human Embryonic Kidney 293T cells (HEK-293T) using the effective drug combinations with respective controls. The biofilm formation and biofilm cell viability were drastically affected with quercetin and selected antibiotics combinations with ≥80% inhibition. In vitro infection studies showed that all the strains could exert significant cell killing (68 to 85%) and the drug combinations decreased the infection rate significantly by reducing the cell killing effect of P. aeruginosa (p<0.05). The synergistic effect of quercetin is attributed to its quorum sensing inhibitory properties. These findings indicate that quercetin along with existing antibiotics can potentiate the treatment against P. aeruginosa infection and may reduce the selection pressure due to antibiotic overuse.

Early detection of leptospirosis using Anti-LipL32 carbon nanotube immunofluorescence probe.

Leptospirosis is a widespread zoonosis and an emerging public health problem. Leptospirosis symptoms are often confused or misdiagnosed with other febrile illness like malaria, viral hepatitis, influenza, dengue, typhoid, melioidosis, and scrub typhus as the clinical manifestations are almost similar. Therefore, early and accurate diagnosis of leptospirosis is indeed critical for proper and prompt treatment. Herein, we report the development of single-walled carbon nanotubes based immunofluorescence probe (Carbo-Lip) for the detection of leptospirosis at an early phase by utilising major outer membrane protein, LipL32 of Leptospira. The Carbo-Lip probe was fabricated through immuno recognition method with fluorescent dye functionalized LipL32 monoclonal antibodies (mAbs), secondary antibody and Leptospira. Surface characterization studies such as Fourier transform infrared spectroscopy with the attenuated total reflectance, scanning electron microscopy, transmission electron microscopy, Zeta potential, and X-ray photoelectron spectroscopy techniques were used to demonstrate the successful fabrication of Carbo-Lip probe. The sensor probe was capable of detecting the presence of leptospires at a lower concentration of 103/ml, and could detect 102 leptospires in 100 µL of sample within 3 h of the test conditions, and was stable up to 2 weeks. This Carbo-Lip probe was further tested and validated for its capacity to detect Leptospira in clinical samples, which exhibited high selectivity and specificity towards Leptospira even in the presence of malaria and dengue. Our results were consistent with microscopic agglutination test, which is known as gold standard, immunoglobulin M (IgM) enzyme-linked immunoassay (ELISA), IgM spot test, and culture tests for the diagnosis of Leptospira infection.

Morphological and micro-tomographic study on evolution of struvite in synthetic urine infected with bacteria and investigation of its pathological biomineralization.

Pathological biomineralization in the urinary system leads to urolithiasis. Formation of kidney stones involves a series of events during which they undergo morphological and mineralogical changes. We investigated the mineralization of biogenic struvite (in vitro) and examined the transformation of distinct interior and exterior structure of struvite. In vitro crystallization of struvite was performed in the presence of two bacteria that were originally isolated from the kidney stone patients. Morphological evaluation was carried out using SR-µCT as well as FESEM, XRD and FT-IR. Characteristic internal 3-D morphology and porosity of the stones were studied. For comparison, patient derived struvite stones were used. From the results obtained, we report that the presence of bacteria enhances the crystallization process of struvite in vitro. A series of time-resolved experiments revealed that struvite crystals experienced a significant morphologic evolution from pin pointed structure to X-shaped and tabular morphologies. These X-shaped and unusual tabular habits of struvite resembled biogenic morphologies of struvite. SR-µCT showed similarities between the patient derived and the in vitro derived struvite crystals. In conclusion, these experiments revealed that the bacteria play a major role in the specific morphogenesis of struvite and can able to control the nucleation, modulate crystalline phases, and shape of the growing crystal.

Anti-quorum sensing potential of Adenanthera pavonina.

BACKGROUND: Quorum sensing (QS) in Pseudomonas aeruginosa plays a key role in virulence factor production, biofilm formation and antimicrobial resistance. Because of emerging antimicrobial resistance in P. aeruginosa, there is a need to find an alternate nonantibiotic agent for the control of infections caused by this organism. OBJECTIVE: To evaluate anti-QS activity of Adenanthera pavonina L., a medicinal plant used in traditional medicine. MATERIALS AND METHODS: Preliminary screening for anti-QS activity of ethanol extract of A. pavonina was carried out using Chromobacterium violaceum CV026 biosensor strain and inhibition of QS-regulated violacein production was quantified using C. violaceum ATCC12472. Bioassay guided fractionation of ethanol extract resulted in ethyl acetate fraction (AEF) with strong anti-QS activity and AEF was evaluated for inhibition of QS-regulated pyocyanin production, proteolytic, elastolytic activity, swarming motility and biofilm formation in P. aeruginosa PAO1. RESULTS: AEF, at 0.5 mg/ml, inhibited pyocyanin production completely and at 1 mg/ml of AEF, complete inhibition of proteolytic and elastolytic activities were observed. However, viability of P. aeruginosa PAO1 was not affected at the tested concentrations of AEF as observed by cell count. Swarming motility was inhibited at the concentration of 0.1 mg/ml of AEF. Thin layer chromatography and biosensor overlay of AEF showed violacein inhibition zone at Rf value 0.63. CONCLUSION: From the results of this study, it can be concluded that A. pavonina extracts can be used as effective anti-QS agents.

Anti-quorum sensing activity of Psidium guajava L. flavonoids against Chromobacterium violaceum and Pseudomonas aeruginosa PAO1.

Psidium guajava L., which has been used traditionally as a medicinal plant, was explored for anti-quorum sensing (QS) activity. The anti-QS activity of the flavonoid (FL) fraction of P. guajava leaves was determined using a biosensor bioassay with Chromobacterium violaceum CV026. Detailed investigation of the effects of the FL-fraction on QS-regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic, elastolytic activities, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 was performed using standard methods. Possible mechanisms of QS-inhibition were studied by assessing violacein production in response to N-acyl homoserine lactone (AHL) synthesis in the presence of the FL-fraction in C. violaceum ATCC31532 and by evaluating the induction of violacein in the mutant C. violaceum CV026 by AHL extracted from the culture supernatants of C. violaceum 31532. Active compounds in the FL-fraction were identified by liquid chromatography-mass spectrometry (LC-MS). Inhibition of violacein production by the FL-fraction in a C. violaceum CV026 biosensor bioassay indicated possible anti-QS activity. The FL-fraction showed concentration-dependent decreases in violacein production in C. violaceum 12472 and inhibited pyocyanin production, proteolytic and elastolytic activities, swarming motility and biofilm formation in P. aeruginosa PAO1. Interestingly, the FL-fraction did not inhibit AHL synthesis; AHL extracted from cultures of C. violaceum 31532 grown in the presence of the FL-fraction induced violacein in the mutant C. violaceum CV026. LC-MS analysis revealed the presence of quercetin and quercetin-3-O-arabinoside in the FL-fraction. Both quercetin and quercetin-3-O-arabinoside inhibited violacein production in C. violaceum 12472, at 50 and 100 µg/mL, respectively. Results of this study provide scope for further research to exploit these active molecules as anti-QS agents.

Inhibition of quorum sensing in Chromobacterium violaceum by Syzygium cumini L. and Pimenta dioica L.

OBJECTIVE: To investigated into the anti-quorum sensing (QS) activity of Syzygium cumini L. (S. cumini) and Pimenta dioica L. (P. dioica) using Chromobacterium violaceum (C. violaceum) strains. METHODS: In this study, anti-QS activity of ethanol extract of Syzygium cumini L. and Pimenta dioica L. were screened using C. violaceum CV026 biosensor bioassay. By bioassay guided fractionation of S. cumini and P. dioica, ethyl acetate fraction (EAF) with strong anti-QS activity was separated. Inhibition of QS regulated violacein production in C. violaceum ATCC12472 by EAF was assessed at different concentrations. The effect of EAF on the synthesis of autoinducer like N-acyl homoserine lactone (AHL) was studied in C. violaceum ATCC31532 using its mutant C. violaceum CV026 by standard methods. RESULTS: EAF inhibited violacein production in C. violaceum ATCC12472 in a concentration dependent manner without significant reduction in bacterial growth. Complete inhibition of violacein production was evidenced in 0.75-1.0 mg/mL concentration of EAF without inhibiting the synthesis of the AHL. TLC biosensor overlay profile of EAF revealed two translucent spots in S. cumini and P. dioica that inhibited C6-AHL mediated violacein production in C. violaceum CV026. CONCLUSIONS: This study indicates the anti-QS activity of the tested medicinal plants against C. violaceum.