Identification of Efflux Pump Mutations in Pseudomonas aeruginosa from Clinical Samples.

Efflux pumps are a specialized tool of antibiotic resistance used by Pseudomonas aeruginosa to expel antibiotics. The current study was therefore conducted to examine the expression of MexAB-OprM and MexCD-OprJ efflux pump genes. In this study, 200 samples were collected from Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC) in Peshawar, Pakistan. All the isolates were biochemically identified by an Analytical Profile Index kit and at the molecular level by Polymerase Chain Reaction (PCR) utilizing specific primers for the OprL gene. A total of 26 antibiotics were tested in the current study using the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and high-level resistance was shown to amoxicillin-clavulanic acid (89%) and low-level to chloramphenicol (1%) by the isolates. The antibiotic-resistant efflux pump genes MexA, MexB, OprM, MexR, MexC, MexD, OprJ, and NfxB were detected in 178 amoxicillin-clavulanic acid-resistant isolates. Mutations were detected in MexA, MexB, and OprM genes but no mutation was found in the MexR gene as analyzed by I-Mutant software. Statistical analysis determined the association of antibiotics susceptibility patterns by ANOVA: Single Factor p = 0.05. The in silico mutation impact on the protein structure stability was determined via the Dynamut server, which revealed the mutations might increase the structural stability of the mutants. The docking analysis reported that MexA wild protein showed a binding energy value of -6.1 kcal/mol with meropenem and the mexA mutant (E178K) value is -6.5 kcal/mol. The mexB wild and mutant binding energy value was -5.7 kcal/mol and -8.0 kcal/mol, respectively. Efflux pumps provide resistance against a wide range of antibiotics. Determining the molecular mechanisms of resistance in P. aeruginosa regularly will contribute to the efforts against the spread of antibiotic resistance globally.

Molecular Characterization and Epidemiology of Antibiotic Resistance Genes of ß-Lactamase Producing Bacterial Pathogens Causing Septicemia from Tertiary Care Hospitals.

Septicemia is a systematic inflammatory response and can be a consequence of abdominal, urinary tract and lung infections. Keeping in view the importance of Gram-negative bacteria as one of the leading causes of septicemia, the current study was designed with the aim to determine the antibiotic susceptibility pattern, the molecular basis for antibiotic resistance and the mutations in selected genes of bacterial isolates. In this study, clinical samples (n = 3389) were collected from potentially infected male (n = 1898) and female (n = 1491) patients. A total of 443 (13.07%) patients were found to be positive for bacterial growth, of whom 181 (40.8%) were Gram-positive and 262 (59.1%) were Gram-negative. The infected patients included 238 males, who made up 12.5% of the total number tested, and 205 females, who made up 13.7%. The identification of bacterial isolates revealed that 184 patients (41.5%) were infected with Escherichia coli and 78 (17.6%) with Pseudomonas aeruginosa. The clinical isolates were identified using Gram staining biochemical tests and were confirmed using polymerase chain reaction (PCR), with specific primers for E. coli (USP) and P. aeruginosa (oprL). Most of the isolates were resistant to aztreonam (ATM), cefotaxime (CTX), ampicillin (AMP) and trimethoprim/sulfamethoxazole (SXT), and were sensitive to tigecycline (TGC), meropenem (MEM) and imipenem (IPM), as revealed by high minimum inhibitory concentration (MIC) values. Among the antibiotic-resistant bacteria, 126 (28.4%) samples were positive for ESBL, 105 (23.7%) for AmpC ß-lactamases and 45 (10.1%) for MBL. The sequencing and mutational analysis of antibiotic resistance genes revealed mutations in TEM, SHV and AAC genes. We conclude that antibiotic resistance is increasing; this requires the attention of health authorities and clinicians for proper management of the disease burden.

Study of variants associated with ventricular septal defects (VSDs) highlights the unique genetic structure of the Pakistani population.

BACKGROUND: Ventricular septal defects (VSDs) are one of the leading causes of death due to cardiac anomalies during the first months of life. The prevalence of VSD in neonates is reported up to 4%. Despite the remarkable progress in medication, treatment and surgical procedure for VSDs, the genetic etiology of VSDs is still in infancy because of the complex genetic and environmental interactions. METHODS: Three hundred fifty subjects (200 VSD children and 150 healthy controls) were recruited from different pediatric cardiac units. Pediatric clinical and demographic data were collected. A total of six variants, rs1017 (ISL1), rs7240256 (NFATc1), rs36208048 (VEGF), variant of HEY2, rs11067075 (TBX5) and rs1801133 (MTHFR) genes were genotyped by tetra-ARMS PCR and PCR-RFLP methods. RESULTS: The results showed that in cases, the rs1017 (g.16138A > T) variant in the ISL1 gene has an allele frequency of 0.42 and 0.58 respectively for the T and A alleles, and 0.75 and 0.25 respectively in the controls. The frequencies of the AA, TA and TT genotypes were, 52%, 11% and 37% in cases versus 21%, 8% and 71% respectively in the controls. For the NFATc1 variant rs7240256, minor allele frequency (MAF) was 0.43 in cases while 0.23 in controls. For the variant in the VEGF gene, genotype frequencies were 0% (A), 32% (CA) and 68% (CC) in cases and 0.0%, 33% and 67% respectively in controls. The allele frequency of C and A were 0.84 and 0.16 in cases and 0.83 and 0.17 respectively in controls. The TBX5 polymorphism rs11067075 (g.51682G > T) had an allelic frequency of 0.44 and 0.56 respectively for T and G alleles in cases, versus 0.26 and 0.74 in the controls. We did not detect the presence of the HEY2 gene variant (g.126117350A > C) in our pediatric cohort. For the rs1801133 (g.14783C > T) variant in the MTHFR gene, the genotype frequencies were 25% (CC), 62% (CT) and 13% (TT) in cases, versus 88%, 10% and 2% in controls. The ISL1, NFATc1, TBX5 and MTHFR variants were found to be in association with VSD in the Pakistani pediatric cohort whilst the VEGF and HEY2 variants were completely absent in our cohort. CONCLUSION: We propose that a wider programme of genetic screening of the Pakistani population for genetic markers in heart development genes would be helpful in reducing the risk of VSDs.

Isolation and molecular characterization of extended spectrum beta lactamase producing Escherichia coli from chicken meat in Pakistan.

The goal of this study was to find E. coli, a prevalent pathogen that causes food-borne illnesses, in chicken samples (n = 500) collected from three districts in KhyberPukhtunkhwa: Mardan, Swabi, and Swat. The E. coli isolates were identified by Gram staining, API strips and Universal Stress Protein. A total of 412 samples tested positive for E. coli and were sensitive to MEM, TZP, and FOS as evidenced by disc diffusion method. The isolates were resistant to TE, NOR, and NA with statistically significant results (P≤0.05). The isolates showed the presence of different antibiotic resistance genes; blaOXA-1, blaCTX-M15, blaTEM-1, QnrS, TetA, AAC, AAD, Sul1 and Sul2. The results revealed mutations in blaOXA-1 gene (H81Q), blaTEM-1 (C108Y, T214A, K284E and P301S), QnrS (H95R) and Sul2 (E66A). The findings of this study may be helpful in better management of E. coli infections by physicians.

Influence of Iron Sulfide Nanoparticle Sizes in Solid-State Batteries*.

Given the inherent performance limitations of intercalation-based lithium-ion batteries, solid-state conversion batteries are promising systems for future energy storage. A high specific capacity and natural abundancy make iron disulfide (FeS2 ) a promising cathode-active material. In this work, FeS2 nanoparticles were prepared solvothermally. By adjusting the synthesis conditions, samples with average particle diameters between 10 nm and 35 nm were synthesized. The electrochemical performance was evaluated in solid-state cells with a Li-argyrodite solid electrolyte. While the reduction of FeS2 was found to be irreversible in the initial discharge, a stable cycling of the reduced species was observed subsequently. A positive effect of smaller particle dimensions on FeS2 utilization was identified, which can be attributed to a higher interfacial contact area and shortened diffusion pathways inside the FeS2 particles. These results highlight the general importance of morphological design to exploit the promising theoretical capacity of conversion electrodes in solid-state batteries.

A High-Performance Asymmetric Supercapacitor Based on Tungsten Oxide Nanoplates and Highly Reduced Graphene Oxide Electrodes.

Tungsten oxide/graphene hybrid materials are attractive semiconductors for energy-related applications. Herein, we report an asymmetric supercapacitor (ASC, HRG//m-WO3 ASC), fabricated from monoclinic tungsten oxide (m-WO3 ) nanoplates as a negative electrode and highly reduced graphene oxide (HRG) as a positive electrode material. The supercapacitor performance of the prepared electrodes was evaluated in an aqueous electrolyte (1 m H2 SO4 ) using three- and two-electrode systems. The HRG//m-WO3 ASC exhibits a maximum specific capacitance of 389 F g-1 at a current density of 0.5 A g-1 , with an associated high energy density of 93 Wh kg-1 at a power density of 500 W kg-1 in a wide 1.6 V operating potential window. In addition, the HRG//m-WO3 ASC displays long-term cycling stability, maintaining 92 % of the original specific capacitance after 5000 galvanostatic charge-discharge cycles. The m-WO3 nanoplates were prepared hydrothermally while HRG was synthesized by a modified Hummers method.

Synthesis of Au, Ag, and Au-Ag Bimetallic Nanoparticles Using Pulicaria undulata Extract and Their Catalytic Activity for the Reduction of 4-Nitrophenol.

Plant extract of Pulicaria undulata (L.) was used as both reducing agent and stabilizing ligand for the rapid and green synthesis of gold (Au), silver (Ag), and gold-silver (Au-Ag) bimetallic (phase segregated/alloy) nanoparticles (NPs). These nanoparticles with different morphologies were prepared in two hours by stirring corresponding metal precursors in the aqueous solution of the plant extracts at ambient temperature. To infer the role of concentration of plant extract on the composition and morphology of NPs, we designed two different sets of experiments, namely (i) low concentration (LC) and (ii) high concentration (HC) of plant extract. In the case of using low concentration of the plant extract, irregular shaped Au, Ag, or phase segregated Au-Ag bimetallic NPs were obtained, whereas the use of higher concentrations of the plant extract resulted in the formation of spherical Au, Ag, and Au-Ag alloy NPs. The as-prepared Au, Ag, and Au-Ag bimetallic NPs showed morphology and composition dependent catalytic activity for the reduction of 4-nitrophenol (4-NPh) to 4-aminophenol (4-APh) in the presence of NaBH4. The bimetallic Au-Ag alloy NPs showed the highest catalytic activity compared to all other NPs.

Pharmacological activities of Justicia adhatoda.

The current study focused on the pharmacological activities of Justicia adhatoda; including antibacterial, antifungal, phytotoxic, cytotoxic, haemagglutination, insecticidal, in vitro antiglycation, DPPH antioxidant and anti-termite. The crude methanolic extract (Crd. Met. Ext) showed 46.4 % antibacterial activity against M. morganii while the n-hexane fraction showed good (71.4%) and moderate (55.1%) activity against M. morganii and A. baumannii respectively. The EtOAc and aqueous fractions, in most of the cases, showed low to no activity against the selected bacterial pathogens, against A. niger, T. harzianum, A. parasiticus and V. dahliae. The Crd. Met. Ext and fractions showed low activity, against P. notatum and P. digitatum, Crd. Met. Ext. and all fractions were inactive. The percent growth regulation, in case of phytotoxic activity, by Crd. Met. Ext was 25 and 16.6, n-hexane fraction 16.6, 16.6 and 0, CHCl3 25, 8.33 and 0 % and EtOAc fractions 8.33, 8.33 and 0% at 1000 and 100 and 10µg/ml respectively. The aqueous fraction was inactive at all the test concentrations. The results of brine shrimp cytotoxic activity for Crd. Met. Ext was 13.33% and n-hexane fraction 20% at 1000, µg/ml respectively. All of the other fractions showed low to no activity at different test concentrations. All of the test samples were inactive against RBC's of the blood groups at all concentration indicating that the selected plant lack phytolectins and haemagglutination activity. The Crd. Met. Ext and various fraction showed low activity against the test insects i.e. C. pulicaria, C. chinensis and T. castaneum. The absorbance value of plant extract for anti-glycation activity at various concentration were: 0.08, 0.067, 0.053 and 0.04 in comparison with Aminoguanidine 0.04, 0.035, 0.03 and 0.02 respectively at 10, 50, 90 and 130µl. The DPPH radical scavenging activities were proportional to the concentration of the fractions, as the concentration of these increased, the percent scavenging activity also increased. The CHCl3 and EtOAc fractions killed all the termites in 24 hours while Crd. Met. Ext, n-hexane and aqueous fractions took 2-3 days.