Selective biodegradation of recalcitrant black chicken feathers by a newly isolated thermotolerant bacterium Pseudochrobactrum sp. IY-BUK1 for enhanced production of keratinase and protein-rich hydrolysates.

Black chicken feathers generated in large amount from poultry and slaughter houses are highly recalcitrant to microbial degradation due to their tough structural nature. A novel keratinolytic bacterium that possessed high affinity for black feather was isolated from chicken manure and identified as Pseudochrobactrum sp. IY-BUK1. Keratinase and feather soluble protein were effectively produced by the free living cells of the bacterium in media containing only black feathers and a mixture of equal amount of black-, brown- and white-coloured feathers. Complete degradation of 5 g/L of black feathers was completed in 3 days following optimisation of physico-chemical conditions. However, the bacterium selectively completed the degradation of black feather in a medium containing mixture of feathers in 144 h leaving behind approximately 33% and 45% of brown and white feathers in the medium respectively. Gellan gum-immobilised cells of strain IY-BUK1 enhanced the keratinase production by about 150% and were used repeatedly for ten cycles to degrade 5 g/L of black feather in a semi continuous fermentation of 18 h per cycle with enhanced and stable production of soluble protein. The study demonstrated the potential use of Pseudochrobactrum sp. IY-BUK1 not only in biodegradation of highly recalcitrant black feathers, but also in producing keratinase enzymes and valuable soluble proteins for possible industrial usage.

Socio-economic determinants of ownership and use of treated bed nets in Nigeria: results from a cross-sectional study in Cross River and Bauchi States in 2011.

BACKGROUND: Poor people bear a disproportionate burden of malaria and prevention measures may not reach them well. A study carried out to examine the socio-economic factors associated with ownership and use of treated bed nets in Cross River and Bauchi States of Nigeria took place soon after campaigns to distribute treated bed nets. METHODS: A cross-sectional household survey about childhood illnesses among mothers of children less than four years of age and focus group discussions in 90 communities in each of the two states asked about household ownership of treated bed nets and their use for children under four years old. Bivariate and multivariate analyses examined associations between socio-economic and other variables and these outcomes in each state. RESULTS: Some 72% of 7,685 households in Cross River and 87% of 5,535 households in Bauchi State had at least one treated bed net. In Cross River, urban households were more likely to possess bed nets, as were less-poor households (enough food in the last week), those with a male head, and those from communities with a formal health facility. In Bauchi, less-poor households and those with a more educated head were more likely to possess nets. In households with nets, only about half of children under four years old always slept under a net: 54% of 11,267 in Cross River and 57% of 11,277 in Bauchi. Factors associated with use of nets for young children in Cross River were less-poor households, fewer young children in the household, more education of the father, antenatal care of the mother, and younger age of the child, while in Bauchi the factors were a mother with more education and antenatal care, and younger age of the child. Some focus groups complained of distribution difficulties, and many described misconceptions about adverse effects of nets as an important reason for not using them. CONCLUSION: Despite a recent campaign to distribute treated bed nets, disadvantaged households were less likely to possess them and to use them for young children. Efforts are needed to reach these households and to dispel fears about dangers of using treated nets.

Cross-sectional survey of knowledge and attitudes of healthcare workers and community members toward the Ebola virus disease and antimicrobial resistance pathogens outbreaks in Nigeria.

INTRODUCTION: the 2014 Ebola virus disease (EVD) outbreak in Nigeria has further raised the awareness of health-care workers (HCWs) and community members (MCs) on the threat posed by infectious diseases and the need for improvement on infection control practices. However, awareness of dangers of increasing incidences of antimicrobial resistance (AMR) in hospitals and communities remained low. METHODS: a cross-sectional survey of awareness of 195 HCWs and 265 MCs toward EVD and AMR was conducted through a structured questionnaire. RESULTS: majority of HCWs (95.4%) and MCs (82.8%) still have knowledge of EVD´s danger and give reasons like its unique way of killing and unavailability of drugs for their awareness. Only 17.2% of MCs are aware of AMR as a problem, and only 3.4% of MCs and 10.3% of HCWs agreed that AMR is more dangerous than EVD. On the contrary, 76.4% of doctors, 95.1% nurses, 67.9% laboratory scientists, 66.7% pharmacists, 77.4% students and 100% of civil servants, drivers and religious leaders believed that EVD is more horrific and spread faster. They both attributed the rapid awareness of EVD in Nigeria, despite being new at the time of the outbreak, to the seriousness with which stakeholders and the media fought EVD, the gesture AMR is yet to receive. Though both HCWs and MCs agreed that prevention, not treatment is the best option to tackle Ebola like-diseases, but surprisingly, about 37% and 65% of HCWs and MCs respectively, still believe that traditional medicines can be used to treat Ebola related illnesses. CONCLUSION: AMR awareness remains low among MCs and some HCWs when compared with EVD. It is recommended that efforts put in place during EVD outbreak by all stakeholders and the media need to be doubled to increase the knowledge of both HCWs and MCs toward AMR.

Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8.

Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with the in vivo activity of the Δ9-fatty acid desaturase on the membrane phospholipids.

Heterologous Expression of PA8FAD9 and Functional Characterization of a Δ9-Fatty Acid Desaturase from a Cold-Tolerant Pseudomonas sp. A8.

Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.

Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3.

Fatty acid desaturase enzymes play an essential role in the synthesis of unsaturated fatty acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- fatty acid desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-fatty acid desaturase capable of increasing the total amount of cellular unsaturated fatty acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-fatty acid desaturase-like protein which was actively expressed in E. coli.