Biocontrol of MRSA and E. coli using bacteriophages from cow manure

Background: Bacteriophages can be used as biocontrol agents to eliminate the undesirable and targeted bacteria in human beings, environment and industries. We aimed to isolate phages from cow manure and investigate their antibacterial and anti-biofilm formation effect against MRSA and E. coli species.Method: E. coli and S. aureus isolates of clinical origin were sub-cultured on MacConkey’s and Mannitol salt agar media, respectively. To confirm the Methicillin resistance in S. aureus, mecA gene was detected by the PCR using gene specific forward and reverse primers. The bacteriophages were isolated using enrichment procedure from cow manure. Their antibacterial and anti-biofilm formation activity was determined through the inhibition of bacterial growth and decrease in the absorbance of Crystal Violet through biofilm inhibition assay, respectively.Results: The isolated phages showed clear plaque formation against MRSA and E. coli species. The time-dependent lytic assay showed a sharp decline in OD600 of MRSA and E. coli after one hour incubation with the phages. Biofilm inhibition was indicated by a decrease in the absorbance (OD595) of CV in a microtiter well plateConclusion: This is perhaps the first study to isolate phages from cow manure at local region and demonstrate their lytic efficiency against MRSA and E. coli, with the ultimate aim to use them as a promising biocontrol agent against antibiotic resistant pathogens.Keywords: Phages; MRSA; Plaque formation; Biofilm inhibition; E. coli; Biocontro

APGAR Score of Neonates Born to Anemic Mothers versus Non-Anemic Mothers

Objective: To determine the effect of maternal iron deficiency anemia on APGAR score of neonates at one minute.Patients and Methods: This cross-sectional study was conducted at Aziz Bhatti Shaheed Teaching Hospital Gujrat from January 2017 to November 2017. In total 380 patients (50% anemic and 50% non-anemic mothers) were included in study. Grouping into anemic and non-anemic groups was done, based on their hemoglobin level, MCV, MCH and ferritin levels. APGAR score at 1 minute was noted in neonates born to both groups. Data was analyzed using SPSS 20.0. Chi square test was applied to evaluate the difference of APGAR score.Results: Mean age in anemic was 27+4.6 years and in non-anemic 26+5.1 years. The mean hemoglobin among anemic was 8.9+2.1 g/dl and among non-anemic 12.3+1.2 g/dL. Mean MCV in non-anemic 85.7+3.8 fl, while among anemic was 62.4+4.1 fl Mean ferritin levels among anemic were 7.4+1.1 ng/ml. Neonates delivered by anemic mothers had mean APGAR score of 6.5+0.2 and those delivered to non-anemic had 8.7+0.5. The APGAR score of neonates delivered by anemic and non-anemic showed a significant difference (p-value 0.00). The relative risk of having low APGAR score of infant in anemic compared to non-anemic mothers was 29.00 (95% CI, 13.1798-63.8098) with p<0.0001.Conclusion: Maternal anemia is significantly associated with poor APGAR score in neonates

Phytochemical screening and assessment of pharmacological properties of Swertia chirayita (Roxb. ex fleming) root methanolic extract

Background and Objective: Various parts of medicinal plants have been used to treat specific disorder from ancient times. Swertia chirayita (Roxb. ex Fleming) is a customary folklore medicine, used in the treatment of liver disorders, fevers, dysentery, diarrhea, stomach problems and other disorders. The present study was carried out in order to assess the antioxidant activity, to evaluate the antifungal properties of the plant’s root and to observe anticancer potential of methanolic extract of Swertia chirayita root. Materials and Methods: Phytochemical analysis and different chemical tests for the screening and identification of bioactive chemical constituents in Swertia chirayita methanolic root extract (SCME) were carried out using the standard procedures. The plants were purchased from local herbal market. In vitro determination of antioxidant properties of SCME were conducted using 1,1-diphenyl-2-picrylhydrazyl (DPPH)radical scavenging activity, H2O2 scavenging activity, Beta-carotene bleaching assay, total antioxidant activity by phosphomolybdenum method, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity and hydroxyl radical scavenging activity. Anticancer activity of SCME was determined according to the protocol of brine shrimp lethality test. Antifungal potential was determined by measuring zone of inhibition on Potato Dextrose Agar (PDA) plates. The different concentrations of SCME used were 3.0, 1.5, 0.75 and 0.37 mg mL–1. The data were evaluated as Mean±Standard Deviations of 5 independent experimental responses. The results were analyzed using t-test for independent samples with SPSS version 16.0. Results: Phytochemical analysis of SCME showed that phlobatannins, tannins, saponins and terpenoids were present. The SCME exhibited strong antioxidant activity in a concentration dependent manner for in all six models. The SCME at dose of 3 mg mL–1 caused 100% death rate of brine shrimp after 72 h. The SCME showed potent activity against Aspergillus flavus (87%) followed by Aspergillus niger (88%) while the highest activity was shown against Aspergillus fumigatus (92%). Conclusion: The SCME exhibited strong antioxidant, antifungal and cytotoxic potential. Purification of different bioactive compounds should be carried out and in vivo studies are required for further verification.Scopu

Determinants of response at 2 months of treatment in a cohort of Pakistani patients with pulmonary tuberculosis

Mycobacterium tuberculosis infection continues to be a major global challenge. All patients with pulmonary tuberculosis are treated with a standard 6-month treatment regimen. Historical data suggest that even with shortened treatment, most patients achieve long-term remission. Risk stratification is a goal for reducing potentially toxic prolonged treatment. This study aimed to determine the factors associated with the early clearance of sputum acid-fast bacilli (AFB). A total of 297 freshly diagnosed patients with pulmonary tuberculosis were included and enrolled in this study. Information related to their ethno-demographic and anthropometric characteristics was collected. We also assessed their complete blood counts, and blood iron, folate, and vitamin B12 levels. We found that the presence of higher levels of acid-fast bacilli (AFB) in diagnostic sputum microscopy was the single most significant prognostic factor associated with early clearance of sputum AFB after 2 months of treatment. All of our patients achieved treatment success after 6 months of treatment and were disease free. Our results support the data obtained from previous studies indicating that AFB clearance at 2 months is unlikely to be a clinically useful biomarker or indicator for therapeutic stratification. Furthermore, demographic, anthropometric, and nutritional factors are not clinically useful biomarkers

Effect of early tranexamic acid administration on mortality, hysterectomy, and other morbidities in women with post-partum haemorrhage (WOMAN): an international, randomised, double-blind, placebo-controlled trial

Background Post-partum haemorrhage is the leading cause of maternal death worldwide. Early administration of tranexamic acid reduces deaths due to bleeding in trauma patients. We aimed to assess the effects of early administration of tranexamic acid on death, hysterectomy, and other relevant outcomes in women with post-partum haemorrhage. Methods In this randomised, double-blind, placebo-controlled trial, we recruited women aged 16 years and older with a clinical diagnosis of post-partum haemorrhage after a vaginal birth or caesarean section from 193 hospitals in 21 countries. We randomly assigned women to receive either 1 g intravenous tranexamic acid or matching placebo in addition to usual care. If bleeding continued after 30 min, or stopped and restarted within 24 h of the first dose, a second dose of 1 g of tranexamic acid or placebo could be given. Patients were assigned by selection of a numbered treatment pack from a box containing eight numbered packs that were identical apart from the pack number. Participants, care givers, and those assessing outcomes were masked to allocation. We originally planned to enrol 15 000 women with a composite primary endpoint of death from all-causes or hysterectomy within 42 days of giving birth. However, during the trial it became apparent that the decision to conduct a hysterectomy was often made at the same time as randomisation. Although tranexamic acid could influence the risk of death in these cases, it could not affect the risk of hysterectomy. We therefore increased the sample size from 15 000 to 20 000 women in order to estimate the effect of tranexamic acid on the risk of death from post-partum haemorrhage. All analyses were done on an intention-to-treat basis. This trial is registered with ISRCTN76912190 (Dec 8, 2008); ClinicalTrials.gov, number NCT00872469; and PACTR201007000192283. Findings Between March, 2010, and April, 2016, 20 060 women were enrolled and randomly assigned to receive tranexamic acid (n=10 051) or placebo (n=10 009), of whom 10 036 and 9985, respectively, were included in the analysis. Death due to bleeding was significantly reduced in women given tranexamic acid (155 [1·5%] of 10 036 patients vs 191 [1·9%] of 9985 in the placebo group, risk ratio [RR] 0·81, 95% CI 0·65–1·00; p=0·045), especially in women given treatment within 3 h of giving birth (89 [1·2%] in the tranexamic acid group vs 127 [1·7%] in the placebo group, RR 0·69, 95% CI 0·52–0·91; p=0·008). All other causes of death did not differ significantly by group. Hysterectomy was not reduced with tranexamic acid (358 [3·6%] patients in the tranexamic acid group vs 351 [3·5%] in the placebo group, RR 1·02, 95% CI 0·88–1·07; p=0·84). The composite primary endpoint of death from all causes or hysterectomy was not reduced with tranexamic acid (534 [5·3%] deaths or hysterectomies in the tranexamic acid group vs 546 [5·5%] in the placebo group, RR 0·97, 95% CI 0·87-1·09; p=0·65). Adverse events (including thromboembolic events) did not differ significantly in the tranexamic acid versus placebo group. Interpretation Tranexamic acid reduces death due to bleeding in women with post-partum haemorrhage with no adverse effects. When used as a treatment for postpartum haemorrhage, tranexamic acid should be given as soon as possible after bleeding onset. Funding London School of Hygiene & Tropical Medicine, Pfizer, UK Department of Health, Wellcome Trust, and Bill & Melinda Gates Foundation

Identification of Calpain 10 Isoforms (b, d, e, f & h) Conserved Regions and Possible Functional Prophecy through Bioinformatics

Calpain 10 is an atypical calpain ubiquitously exist in all human tissues. It exhibits eight protein isoforms designated as “a-h” which play a vital role in glucose homeostasis but actual mechanism of action is yet to be ascertained. We have predicted the partial roles of Isoform a, c and g previously. They were envisaged to act partially as mu and m-calpain cysteine proteases. Here we predict the function of minor isoforms b, d, e, f and h. We have applied NCBI Blast and Conserved domain tool for nucleotide and protein alignments. Blast query indicated 87%, 84%, 87%, 94% and 34% identity of isoform b, d, e, f and h with canonical sequence of calpain 10 a isoform. Conserved domain analyses of protein sequences revealed significant structural similarities of their N-terminal domain I and II with catalytic domain of cysteine protease superfamily PC1 (e-value:CAPN10b, d, e = 2.41e-76, CAPN10f = 1.07e-43 and CAPN10h = 1.13e-17). Isoform b, d and e have one consecutive domain similar with C2 like subdomain III (e-value=2.92-32, 1.03e-35, 1.88e-14 respectively) and was classified in CAPN10 group of Palb subfamily. Isoform f and h were lacking this domain and had shorter sequences. Although structural similarities are not guaranteed for similar actions but domain homology predicted the existence of similar functions as of calpain I and II

Clinical profile and screening of exon 6 and 14 of ABCB4 gene in obstetric cholestasis patients at a tertiary care hospital in Rawalpindi, Pakistan

Objective: Intrahepatic Cholestasis of Pregnancy (ICP) is a rare pregnancy specific disorder. Genetic variants of ABCB4 gene increase ICP risk. This study was conducted to determine frequency of ICP cases presented at a tertiary care hospital in Rawalpindi, Pakistan and to screen for genetic variants of exon 6 and 14 of ABCB4 gene inICP cases. Methods: This analytical study included ICP patients presented at Department of Gynaecology and Obstetrics, Holy Family Hospital Rawalpindi, from February 2017 to May 2017. Sanger’s sequencing was performed using genomic DNA extracted from blood samples of patients and controls. Results: Twenty pregnant women out of 1150 (1.74%) had ICP and enrolled during study period. Overall 95% patients had pruritus and among them 40%, 20% and 10% had a history of miscarriages, stillbirths and familial ICP respectively. Genetic analysis revealed an already reported variant i.e., c.504C>T in exon 6 in thirteen patients and a novel variant i.e., c.1686A>G in exon 14 in five patients. Both variants were not present in controls. In silico analysis suggested that both variants might affect pre-mRNA splicing of ABCB4 transcript. Conclusion: ICP had a frequency of 1.74% among pregnant women. Identification of a novel heterozygous variant in five patients and an already reported variant in thirteen patients reaffirms genetic heterogeneity and role of ABCB4 in ICP etiology. Keywords: intrahepatic cholestasis of pregnancy, ABCB4 gene, Single Nucleotide Polymorphism (SNP), Continuous..

Potential of Azotobacter vinelandii Khsr1 as bio-inoculant

The present study deals with the isolation and characterization of Azotobacter vinelandii Khsr1 from roots of the weed, Chrysopogon aucheri, commonly known as golden beard grass indigenous to Khewra salt range, Pakistan and its evaluation as bio-inoculant. The population of the isolate varied from 107 to 108 cfu/g fresh weight of root. The preliminary identification of the isolate was made on the basis of carbon/nitrogen source utilization pattern as revealed by QTS-24 miniaturized identification system test which placed the isolate to the genus Azotobacter. The 16S-rRNA partial sequence analysis confirmed the isolate as A. vinelandii strain Khsr1. The 556 long nucleotide sequence of the isolate showed 98% similarity with A. vinelandii DJ (accession no. 012560.1). The isolate was capable of producing phytohormones: indole-3-acetic acid, gibberellic acid, trans-zeatin riboside and abscisic acid in culture supernatant, stimulated growth of Zea mays L. seedlings and augmented proline content of roots and shoots both under normal and NaCl stressed conditions. However, the magnitude of stimulation is higher under un-stressed condition.Key words: Azotobacter vinelandii Khsr1, phytohormones production, growth stimulation, salinity stress

Isolation and Molecular Characterization of a Model Antagonistic Pseudomonas aeruginosa Divulging In Vitro Plant Growth Promoting Characteristics

The use of microbial technologies in agriculture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. In the present study 18 strains of Pseudomonas were isolated from soil sample of Balochistan coastline. Among isolated Pseudomonas strains four designated as SP19, SP22, PS24, and SP25 exhibited biocontrol activities against phytopathogenic fungi, that is, Rhizopus microsporus, Fusarium oxysporum, Aspergillus niger, Alternaria alternata, and Penicillium digitatum; PS24 identified as Pseudomonas aeruginosa by 16srRNA gene bank accession number EU081518 was selected on the basis of its antifungal activity to explore its potential as plant growth promotion. PS24 showed multiple plant growth promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA), siderophore, and HCN production. In order to determine the basis for antifungal properties, antibiotics were extracted from King B broth of PS24 and analyzed by TLC. Pyrrolnitrin antibiotic was detected in the culture of strain PS24. PS24 exhibited antifungal activities found to be positive for hydrogen cyanide synthase Hcn BC gene. Sequencing of gene of Hcn BC gene of strain PS24 revealed 99% homology with the Pseudomonas aeruginosa strain PA01. The sequence of PS24 had been submitted in gene bank accession number KR605499. Ps. aeruginosa PS24 with its multifunctional biocontrol possessions can be used to bioprotect the crop plants from phytopathogens