Comparative analysis of different methods used for molecular characterization of Burkholderia cepacia complex isolated from noncystic fibrosis conditions

PurposeBurkholderia is a Gram-negative opportunistic bacterium capable of causing severe nosocomial infections. The aim of this study was to characterize Burkholderia cepacia complex and to compare different molecular methods used in its characterization.MethodsIn this study, 45 isolates of Burkholderia cepacia complex (Bcc) isolated from clinical cases were subjected to RAPD (Random amplified polymorphic DNA), recA-RFLP (Restriction fragment length polymorphism), 16SrDNA-RFLP, whole-cell protein analysis, recA DNA sequencing and biofilm assay.ResultsOf the 45 isolates tested, 97.7% were sensitive to ceftazidime, 82.2% were sensitive to Cotrimoxazole, 73.3% were sensitive to meropenem, 55.5% were sensitive to minocycline and 42.2% were sensitive to levofloxacin. Majority of the isolates harbored all the tested virulence genes except bpeA and cblA. The RAPD generated 11 groups (R1-R11), recA-RFLP 10 groups (A1-A10), 16SrRNA-RFLP 5 groups (S1–S5) and SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) whole cell protein analysis revealed 12 groups (C1–C12). recA sequencing revealed that most of the isolates belonging to the genomovar III Burkholderia cenocepacia. Though all the methods are found to be efficient in differentiating Burkholderia spp., recA-RFLP was highly discriminatory at 96% similarity value. The study also identified a new strain Burkholderia pseudomultivorans for the first time in the country. Further, recA sequencing could identify the strains to species level. Majority of the multidrug-resistant strains also showed moderate to strong biofilm-forming ability, which further contributes to the virulence characteristics of the pathogens.ConclusionsThe study highlights the importance of combination of molecular methods to characterize Burkholderia cepacia complex. Molecular typing of these human pathogens yields important information for the clinicians in order to initiate the most appropriate therapy in the case of severe infections and to implement preventive measures for the effective control of transmission of Burkholderia spp.     

Engineering of polymer–surfactant nanoparticles of doxycycline hydrochloride for ocular drug delivery

Abstract

Context: Physiologic barriers of the eye, short precorneal drug residence time and poor corneal penetration are the few reasons for reduced ocular bioavailability.

Objective: This study was aimed to develop novel polymer–surfactant nanoparticles of hydrophilic drug doxycycline hydrochloride (DXY) to improve precorneal residence time and drug penetration.

Materials and methods: Nanoparticles were formulated using emulsion cross-linking method and the formulation was optimized using factorial design. The prepared formulation was characterized for particle size, ζ potential, encapsulation efficiency, in vitro drug release and ex vivo drug diffusion studies. The antibacterial activity studies were also carried out against Escherichia coli and Staphylococcus aureus using the cup-plate method. In vivo eye irritation study was carried out by a modified Draize test in rabbits.

Results and discussion: The particle size was found to be in the range of 331–850?nm. About 45–80% of the drug was found to be encapsulated in the nanoparticles. In vitro release demonstrated sustained release profile. Lower flux values in case of nanoparticles as compared to DXY pure drug solution in ex vivo diffusion studies confirmed the sustained release. The nanoparticles were found to be significantly effective (p?<?0.001) than DXY aqueous solution due to sustained release of doxycycline from nanoparticles in both the E. coli and S. aureus strains. The formulation was found to be stable over entire stability period.

Conclusion: The developed formulation is safe and suitable for sustained ocular drug delivery.     

Structure–Function Analysis of Liver Flavin Monooxygenase 3 that Drives Trimethylaminuria in Humans

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Mutations in human flavin monooxygenase-3 (hFMO3) enzyme have been implicated in the rare autosomal recessive...     

Keratoglobus: An experience at a tertiary eye care center in India

Context:

This study was carried out as a part of an internal audit and is the largest series of patients having keratoglobus, published in the literature. Poor visual acuity of the patients indicates the blinding nature of the disease.

Aims:

We report our experience with patients having keratoglobus at a tertiary eye care center in India.

Settings and Design:

Retrospective study.

Materials and Methods:

We analyzed adults and pediatric patients (<16 years) with keratoglobus, seen during 2008–2012. The age, gender, consanguinity, presenting ocular signs, ocular and systemic associations, visual acuity, corneal topography, and surgeries were documented.

Results:

Forty-eight patients (mean age 22 ± 15 years, 31 males) having keratoglobus were analyzed. 21 patients (42 eyes) were <16 years. Twelve eyes (16 events) had positive history of trauma. The presenting clinical signs were corneal scars/scars of tear repair (15 eyes), hydrops, healed and acute (14 eyes) and corneal or globe rupture (9 eyes). Best-corrected visual acuity was >20/40 in 6/42 (14.3%) pediatric eyes and 15/53 (28.30%) adults. Visual acuity ranging from counting of fingers to no light perception was noted in 20/53 (37.74%) adults and 21/42 (50%) pediatric patients; 13/20 (65%) with blue sclera and 8/22 eyes (36.37%) without blue sclera. Vernal keratoconjunctivitis was present in one pediatric patient. Choroidal osteoma, retinitis pigmentosa, and retinal detachment were present in adults. Surgeries performed were corneal tear repair (5 eyes), tissue adhesive application (2 eyes), descematopexy (4 eyes) and penetrating keratoplasty (PK - 8 eyes: Three had post-PK glaucoma, graft failure-one eye, 4 patients wore scleral lens - prosthetic replacement of the ocular surface ecosystem).

Conclusions:

About 50% of pediatric eyes (65% having blue sclera) had no functional vision. Trivial trauma was responsible for corneal rupture indicating need for protective glasses. About 50% patients had post-PK glaucoma though grafts were clear.