Phenotypic Characterization and Whole-Genome Analysis of a Novel Bacteriophage HCF1 Infecting Citrobacter amalonaticus and C. freundii.

Citrobacter species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. Citrobacter spp. cause urinary tract infections (UTIs) and infantile meningitis in humans. Due to the presence of plasmid-encoded resistance genes, Citrobacter spp. are often resistant to many antibiotics. In this study, Citrobacter virus HCF1, a novel virulent bacteriophage capable of killing Citrobacter amalonaticus and Citrobacter freundii, was isolated from the sewage water. The isolated bacteriophage was characterized with respect to transmission electron microscopy, one-step growth curve, host range, in vitro efficacy, storage stability, and environmental stress tolerance. The one-step growth curve analysis revealed that the latent period of HCF1 was 30 min and the estimated burst size was 121 plaque-forming units (PFU) per bacterial cell. Host range testing indicated that the HCF1 was specific to the Citrobacter genus. In vitro efficacy assay in the effluent of an anaerobic biodigester showed that the HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively, thereby indicating its potential for combating C. amalonaticus infections. The isolated bacteriophage is considerably stable and tolerant to environmental stress. Furthermore, the complete genome of HCF1 was sequenced using Oxford Nanopore sequencing and the data were subjected to detailed bioinformatic analyses. NCBI-BLASTn analysis revealed that the HCF1 genome had a query coverage of 15-21% and a maximum similarity of 77.27-78.49% with 11 bacteriophages of the Drexlerviridae family. Detailed bioinformatic analysis of the genome profile suggests that HCF1 is a novel T1svirus belonging to the Tempevirinae subfamily of the Drexlerviridae family.

Comparison of acid and water-soluble chitosan doped fibrous cellulose hemostat wet laid nonwoven web for hemorrhage application.

Fibrous hemostat was produced using wet laying technique comprising of acid and water soluble chitosan with fibrous cellulose. The hemostatic efficiency and their structural properties were evaluated using blood clotting time, whole blood adhesion and analytical characterization techniques. The maximum concentration of chitosan utilised for the production of nonwoven was 1.5 w/v % upon which increased the viscosity of the solution. The absorption of phosphate buffer solution was found to be 1117% for acid soluble chitosan based nonwoven whereas for water soluble chitosan based nonwoven was 997%. Increased red blood cells and platelets were found to be higher in acid soluble chitosan compared to water soluble chitosan based cellulose nonwoven. The blood clotting time for acid soluble chitosan nonwoven was found to be 166 s and water soluble chitosan nonwoven was 170 s. The developed hemostat have potential application in reducing blood loss and also inducing the blood coagulation pathway which was confirmed by the adhesion of platelets on to the surface of nonwoven web.

Porous electrospun starch rich polycaprolactone blend nanofibers for severe hemorrhage.

Starch nanofiber based hemostat was prepared by electrospinning process for biomedical applications. Bead free uniform starch nanofiber along with polycaprolactone polymer was produced using new solvent combination. The swelling of the PCL/starch mat was 240% higher compared to pristine PCL mat. The blood clotting time of the developed PCL/starch mat was 156 s and the contact angle was 30.8°. The results suggest that the developed nanofibers exhibited good hemostatic potential with quick rate of clotting to control blood loss in traumatic injuries.

Biology & control of Anopheles culicifacies Giles 1901.

Malaria epidemiology is complex due to multiplicity of disease vectors, sibling species complex and variations in bionomical characteristics, vast varied terrain, various ecological determinants. There are six major mosquito vector taxa in India, viz. Anopheles culicifacies, An. fluviatilis, An. stephensi, An. minimus, An. dirus and An. sundaicus. Among these, An. culicifacies is widely distributed and considered the most important vector throughout the plains and forests of India for generating bulk of malaria cases (>60% annually). Major malaria epidemics are caused by An. culicifaices. It is also the vector of tribal malaria except parts of Odisha and Northeastern States of India. An. culicifacies has been the cause of perennial malaria transmission in forests, and over the years penetrated the deforested areas of Northeast. An. culicifacies participates in malaria transmission either alone or along with An. stephensi or An. fluviatilis. The National Vector Borne Disease Control Programme (NVBDCP) spends about 80 per cent malaria control budget annually in the control of An. culicifacies, yet it remains one of the most formidable challenges in India. With recent advances in molecular biology there has been a significant added knowledge in understanding the biology, ecology, genetics and response to interventions, requiring stratification for cost-effective and sustainable malaria control. Research leading to newer interventions that are evidence-based, community oriented and sustainable would be useful in tackling the emerging challenges in malaria control. Current priority areas of research should include in-depth vector biology and control in problem pockets, preparation of malaria-risk maps for focused and selective interventions, monitoring insecticide resistance, cross-border initiative and data sharing, and coordinated control efforts for achieving transmission reduction, and control of drug-resistant malaria. The present review on An. culicifacies provides updated information on vector biology and control outlining thrust areas of research.

Status of DDT and pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance (kdr) mutation.

BACKGROUND & OBJECTIVES: Aedes albopictus is one of the vectors for dengue and chikungunya and emergence of pyrethroid resistance in this species could be of a major concern in controlling the vector. This study reports insecticide susceptibility status of Ae. albopictus to DDT and pyrethroids in some Indian populations and status of presence of knockdown resistance (kdr) mutations. METHODS: Three to four day old adult female Ae. albopictus collected from Delhi, Gurgaon (Haryana), Hardwar (Uttarakhand), Guwahati (Assam) and Kottayam (Kerala) were bio-assayed with DDT (4%), permethrin (0.75%) and deltamethrin (0.05%) impregnated papers using WHO standard susceptibility test kit. Mosquitoes were PCRgenotyped for F1534C kdr-mutation in the voltage-gated sodium channel (VGSC) gene. DDT and pyrethroid resistant individuals were sequenced for partial domain II, III and IV of VGSC targeting residues S989, I1011, V1016, F1534 and D1794 where kdr mutations are reported in Ae. aegypti. RESULTS: Adult bioassays revealed varying degree of resistance against DDT among five populations of Ae. albopictus with corrected mortalities ranging between 61 and 92%. Kerala and Delhi populations showed incipient resistance against permethrin and deltamethrin respectively. All other populations were susceptible for both the synthetic pyrethroids. None of the kdr mutations was detected in any of DDT, deltamethrin and permethrin resistant individuals. INTERPRETATION & CONCLUSION: Ae. albopictus has developed resistance against DDT and there is emergence of incipient resistance against pyrethroids in some populations. So far, there is no evidence of presence of knockdown resistance (kdr) mutation in Ae. albopictus.

Dengue, an emerging arboviral infection in Assam, northeast India.

Dengue is emerging as major public health concern in northeast India and spreading with increased morbidity. Most cases were recorded in Guwahati metropolitan city of the state of Assam during post-monsoon months (September- December). These comprised all age groups of both sexes with significantly higher incidence of cases in adult males aged 26- 60 years.

Mosquito-borne diseases in Assam, north-east India: current status and key challenges.

Mosquito-borne diseases, including malaria, Japanese encephalitis (JE), lymphatic filariasis and dengue, are major public health concerns in the north-eastern state of Assam, deterring equitable socioeconomic and industrial development. Among these, malaria and JE are the predominant infections and are spread across the state. The incidence of malaria is, however, gradually receding, with a consistent decline in cases over the past few years, although entry and spread of artemisinin-resistant Plasmodium falciparum remains a real threat in the country. JE, formerly endemic in upper Assam, is currently spreading fast across the state, with confirmed cases and a high case-fatality rate affecting all ages. Lymphatic filariasisis is prevalent but its distribution is confined to a few districts and disease transmission is steadily declining. Dengue has recently invaded the state, with a large concentration of cases in Guwahati city that are spreading to suburban areas. Control of these diseases requires robust disease surveillance and integrated vector management on a sustained basis, ensuring universal coverage of evidence-based key interventions based on sound epidemiological data. This paper aims to present a comprehensive review of the status of vector-borne diseases in Assam and to address the key challenges.

Naturally derived biofunctional nanofibrous scaffold for skin tissue regeneration.

Significant wound healing activity of Aloe vera (AV) and higher elastic strength of Silk fibroin (SF) along with mammalian cell compatibility makes AV and SF an attractive material for tissue engineering. The purpose of the present work was to combine their unique properties, with the advantage of electrospinning to prepare a hybrid transdermal biomaterial for dermal substitutes. The physico-chemical characterization of the developed scaffold showed finer morphology expressing amino and esteric groups with improved hydrophilic properties and favorable tensile strain of 116% desirable for skin tissue engineering. Their biological response showed favorable fibroblast proliferation compared to control which almost increased linearly by (p<0.01) 34.68% on day 3, (p<0.01) 19.13% on day 6, and (p<0.001) 97.86% on day 9 with higher expression of CMFDA, collagen and F-actin proteins. The obtained results prove that the nanofibrous scaffold with synergistic property of AV and SF would be a potential biomaterial for skin tissue regeneration.

Dengue vectors in urban and suburban Assam, India: entomological observations.

BACKGROUND: Dengue is rapidly becoming established in north-east India and spreading, on account of rapid urbanization and population movement, with reported morbidity and attributable death cases. This study aims to determine the seasonal abundance of Aedes (Stegomyia) albopictus and Aedes (Stegomyia) aegypti in Guwahati metropolis and suburban settlements; to characterize the breeding resources for these mosquitoes; and to ascertain the status of their susceptibility to adulticides and larvicides. METHODS: Mosquito larval surveys were carried out in different localities in both Guwahati city and adjoining suburbs from January to December 2013, to determine the seasonal abundance of disease vectors and their breeding preferences. The insecticide susceptibility status of mosquito adults and larval populations of both Aedes aegypti and Aedes albopictus was ascertained, using World Health Organization standard diagnostic concentrations and test procedures. RESULTS: The study revealed that both Aedes aegypti and Aedes albopictus are widely abundant in Guwahati city and suburbs, and breeding in a wide variety of resources. Aedes albopictus, however, was the predominant mosquito species in suburbs, breeding preferentially in flower vases, cut-bamboo stumps and leaf axils. Aedes aegypti was the most common in the city, breeding predominantly in discarded tyres, cement tanks and used battery boxes. Both Aedes aegypti and Aedes albopictus were resistant to dichlorodiphenyltrichloroethane (DDT; 4%), but susceptible to malathion (5%), and exhibited a varied response to pyrethroids. However, larval populations of both these mosquito species were susceptible to larvicides, including malathion (1.0 mg/L), temephos (0.02 mg/L) and fenthion (0.05 mg/L), at much lower dosages than diagnostic concentrations. CONCLUSION: Given the seasonal abundance and case incidence in city areas, it is highly probable that Aedes aegypti is the predominant mosquito vector transmitting dengue virus. The study results have direct relevance for the state dengue-control programme, for targeting interventions and averting outbreaks and spread of disease.

Electrosprayed hydroxyapatite on polymer nanofibers to differentiate mesenchymal stem cells to osteogenesis.

Electrospraying of hydroxyapatite (HA) nanoparticles onto the surface of polymer nanofibers provides a potentially novel substrate for the adhesion, proliferation and differentiation of mesenchymal stem cells (MSCs) into bone tissue regeneration. HA nanoparticles (4%) were electrosprayed on the surface of electrospun polycaprolactone (PCL) nanofibers (420 ± 15 nm) for bone tissue engineering. PCL/HA nanofibers were comparatively characterized with PCL/Collagen (275 ± 56 nm) nanofibers by FT-IR analysis to confirm the presence of HA. Fabricated PCL/HA and PCL/Collagen nanofibers and TCP (control) were used for the differentiation of equine MSC into osteogenic lineages in the presence of DMEM/F12 medium supplemented with ß-glycerophosphate, ascorbic acid and dexamethasone. Cell proliferation and differentiation into an osteogenic lineage was evaluated by MTS assay, SEM observation, ALP activity, ARS staining, quantification of mineral deposition and expression of osteocalcin. Proliferation of MSCs increased significantly (P ⩽ 0.05) up to 12% in PCL/Collagen (day 15) compared to PCL/HA nanofibrous substrate. ALP activity was increased 20% in PCL/HA by day 10 confirming the direction of osteogenic lineage from MSCs differentiation. PCL/HA stimulated an increased mineral secretion up to 26% by day 15 on ARS staining compared to PCL/Collagen nanofibers and showing cuboidal morphology by expressing osteocalcin. These results confirmed that the specifically fabricated PCL/HA composite nanofibrous substrate enhanced the differentiation of MSCs into osteogenesis.

Laboratory wash-resistance and field evaluation of deltamethrin incorporated long-lasting polyethylene netting (Netprotect(®)) against malaria transmission in Assam, north-east India.

North-east India is co-endemic for Plasmodium falciparum and P. vivax malaria, and disease transmission is perennial and persistent. This study reports the results of a field-based village scale trial of deltamethrin incorporated long-lasting polyethylene netting (Netprotect(®)) conducted in P. falciparum predominant pocket of Assam, north-east India to assess operational feasibility, acceptability and sustainability against disease vectors and malaria transmission. The study monitored the residual efficacy of the long-lasting net in relation to serial washings in the laboratory and malaria prevalence in experimental villages for the first year of investigations from September 2008 to June 2009. The mosquito vector populations of Anopheles minimus were observed to be highly susceptible to deltamethrin (0.05%), and follow up investigations revealed that the vector mosquito had virtually disappeared in Netprotect(®) intervention villages. Concurrently, there was consistent decline in malaria cases in Netprotect(®) villages and transmission reduction was statistically significant compared to untreated net (net without insecticide) and no-net control villages for the corresponding study period. The contact cone-bioassay investigations against malaria transmitting mosquito species revealed that the bioavailability of the insecticide on the net fiber was persistent up to 20th serial wash resulting in ≥80% mortality. Community compliance and acceptance were high, and users reported decreased nuisance due to biting mosquitoes. It was concluded that deltamethrin incorporated polyethylene long-lasting netting was safe, wash-resistant, and assessed to be an operationally feasible, community-based intervention for sustainable management of disease vectors to prevent malaria transmission.

Primary congenital glaucoma associated with Patau syndrome with long survival.

Ocular abnormalities are common in Patau syndrome (trisomy 13), but only a few cases with congenital glaucoma have been reported, some of which were associated with other ocular defects. This report describes a case of primary congenital glaucoma in an 11-year-old patient with full trisomy 13.

Wash resistance and residual efficacy of long-lasting polyester netting coated with alpha-cypermethrin (Interceptor) against malaria-transmitting mosquitoes in Assam, northeast India.

Malaria is endemic in Assam, northeast India, with low-to-moderate transmission of the causative parasites, mostly by Anopheles minimus. Plasmodium falciparum is the predominant parasite (>60%), with remaining cases being due to P. vivax. As an alternative intervention for malaria control, long-lasting insecticidal nets [Interceptor coated with alpha-cypermethrin 10% suspension concentrate (SC), 0.667% w/w, 0.2g/m(2)] underwent field evaluation for laboratory wash resistance and residual efficacy in field conditions against malaria-transmitting mosquitoes. Based on entomological observations, the Interceptor net intervention was the most effective, corresponding to the lowest mosquito vector density in experimental villages. There was virtual disappearance of A. minimus in Interceptor net villages in contrast to the untreated net intervention and the no-net control. Contact cone bioassay tests revealed 100% mortality in the A.minimus group of mosquito species in the community using the Interceptor net, which was consistent during the follow-up monitoring period (October 2006 to April 2007) in field conditions. Similar levels of mortality were observed in laboratory-washed nets compared with unwashed nets, and wash resistance was consistent even after the 20th serial wash at fortnightly intervals. Community compliance and acceptance of the Interceptor net was high, with decreased nuisance due to biting mosquitoes and other household insect pests being reported.

Continuous nanostructures for the controlled release of drugs.

The annual world wide market for controlled release of polymer systems which extends beyond drug delivery is now estimated to $60 billion and these systems are used by over 100 million people each year. It was estimated that drug delivery will play a pivotal role in approximately 40% of all pharmaceutical sales in near future. Novel methods of drug delivery will not only result in more effective and efficacious treatments but also generates new niche markets to provide greater intellectual property protection to already existing drug formulations. Recently, biodegradable electrospun polymer nanofibrous substrate as drug carrier seems to be a promising method for delivering anticancer drugs, especially in postoperative local chemotherapy. Alternatively drug release can be triggered by the environment or other external events such as changes in pH, temperature, or the presence of analyte such as glucose. In general, controlled release of polymer systems delivering drugs in the optimum dosage for long periods is to increase the efficacy of drug, reducing patient compliance. Recent research for the use of nanotechnology (nanoparticle and nanofibers) in drug delivery suggests that the technology might solve problems in the areas such as controlled release, various topical administration, gut absorption and targeted systemic delivery. This review article described the applications of polymer nanoparticles and nanofibers for loading potential drugs for the controlled release to target incurable diseases.

Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue engineering.

The current challenge in peripheral nerve tissue engineering is to produce an implantable scaffold capable of bridging long nerve gaps that will produce results similar to autograft without requiring the harvest of autologous donor tissue. Aligned and random polycaprolactone/gelatin (PCL/gelatin) nanofibrous scaffolds were fabricated for the in vitro culture of Schwann cells that assist in directing the growth of regenerating axons in nerve tissue engineering. The average fiber diameter attained by electrospinning of polymer blend (PCL/gelatin) ranged from 232+/-194 to 160+/-86nm with high porosity (90%). Blending PCL with gelatin resulted in increased hydrophilicity of nanofibrous scaffolds and yielded better mechanical properties, approaching those of PCL nanofibers. The biocompatibility of fabricated nanofibers was assessed for culturing and proliferation of Schwann cells by MTS assay. The results of the MTS assay and scanning electron microscopy confirmed that aligned and random PCL/gelatin nanofibrous scaffolds are suitable substrates for Schwann cell growth as compared to PCL nanofibrous scaffolds for neural tissue engineering.

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts.

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(epsilon-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200-700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell-scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.

Characterization of Vibrio cholerae from deep ground water in a cholera endemic area in Central India.

A total of 8 out of 11 deep ground water samples collected from different villages in Central India were found contaminated with Vibrio cholerae non O1, non O139. In a multiplex PCR, isolates were found positive for ompW gene but negative for ctxAB and rfbO1 genes. However, isolates from two places were positive for tcp and zot genes, indicating their intestinal colonization and toxigenic potential. Antibiotic susceptibility studies revealed that all isolates were multidrug resistant. Although, none of the isolates was found PCR positive for the mobile genetic elements, class 1 integrons and SXT constins. The results of this study corroborated that deep ground water can also be an important reservoir of V. cholerae in plane endemic areas, suggesting a continuous monitoring of water samples for timely prevention of the disease.

Malaria transmission and disease burden in Assam: challenges and opportunities.

Malaria is major public health illness in Assam and 30-40% of the population is estimated to be at high-risk. Despite decades of attempted control interventions, malaria transmission is perennial and persistent in most parts of the state mostly transmitted by Anopheles minimus. Malaria outbreaks are returning associated with high rise in Plasmodium falciparum and attributable death cases. Therapeutic efficacy investigations for treatment of malaria revealed that chloroquine resistance was widespread for which artemisinin-based combination therapy (ACT) is being instituted in the control program. For data based on the preceding years, we briefly reviewed the available information on transmission dynamics, vector biology and control, drug policy, and discuss the challenges and opportunities for strengthening interventions for malaria control to help design situation specific strategies to check impending disease outbreaks with special reference to Assam. Under increased assistance from external agencies, we strongly advocate scaling up interventions based on mass distribution of long-lasting insecticidal nets (LLINs) for prevention and ACTs for treatment of drug-resistant malaria, and developing strong health delivery system in high-risk areas for meeting the complex emergencies and achieving transmission reduction.