Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli.

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

In vitro study: methylene blue-based antibacterial photodynamic inactivation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most antibiotic-resistant and opportunistic pathogens in immunocompromised and debilitated patients. It is considered the cause of most severe skin infections and is frequently found in hospital burn units. Due to its high antibiotic resistance, eliminating P. aeruginosa from skin infections is quite challenging. Therefore, this study aims to assess the novel in vitro antibacterial activity of methylene blue using a 635-nm diode laser to determine the effective power and energy densities for inhibition of P. aeruginosa. The strain was treated with various concentrations of methylene blue and 635-nm diode laser at powers of 300 mW/cm2 and 250 mW/cm2. The diode laser's potency in the photo-destruction of methylene blue and its degradation through P. aeruginosa were also evaluated. Colony-forming unit (CFU)/ml, fluorescence spectroscopy, optical density, and confocal microscopy were used to measure the bacterial killing effect. As a result, the significant decrease of P. aeruginosa was 2.15-log10, 2.71-log10, and 3.48-log10 at 60, 75, and 90 J/cm2 after excitation of MB for 240, 300, and 360 s at a power of 250 mW/cm2, respectively. However, a maximum decrease in CFU was observed by 2.54-log10 at 72 J/cm2 and 4.32-log10 at 90 and 108 J/cm2 after 300 mW/cm2 of irradiation. Fluorescence images confirmed the elimination of bacteria and showed a high degree of photo-destruction compared to treatment with methylene blue and light alone. In conclusion, MB-induced aPDT demonstrated high efficacy, which could be a potential approach against drug-resistant pathogenic bacteria. KEY POINTS: • Combination of methylene blue with 635-nm diode laser for antibacterial activity. • Methylene blue photosensitizer is employed as an alternative to antibiotics. • aPDT showed promising antibacterial activity against Pseudomonas aeruginosa.

Identification, Genome Sequencing, and Characterizations of Helicobacter pylori Sourced from Pakistan.

The stomach's colonization by Helicobacter pylori (H. pylori) results in gastritis, ulcers, and stomach cancer. Frequently, pain is treated with medication, but resistant H. pylori infections are not. Therefore, it is important to find pharmacological targets and improved treatments for resistant H. pylori strains. The aim of the current study was sampling, identification, drug susceptibility testing following genome sequencing and comparative genome-wide analysis of selected H. pylori strains from Pakistan with three representative strains for virulence and drug-resistant characteristics. Based on culture, biochemistry, and molecular biology, 84 strains of H. pylori were identified, which made up 47% of the enrolled cases. Among all H. pylori strains, the highest resistance was reported for metronidazole with 82 H. pylori strains (98%), followed by clarithromycin with 62 resistant strains (74%). Among metronidazole-resistant strains, 38 strains (46%) were also resistant to clarithromycin, contributing 61% of clarithromycin resistant cases. Two strains, HPA1 and HPA2, isolated from 'gastritis' and 'gastric ulcer' patients, respectively, were further processed for WGS. The draft genome sequences of H. pylori strains HPA1 and HPA2 encode 1.66 Mbp and 1.67 Mbp genome size, 24 and 4 contiguous DNA sequences, and 1650 and 1625 coding sequences, respectively. Both the genomes showed greater than 90% similarity with the reference strain H. pylori ATCC 43504/PMSS1. The antibiotic-resistant genes were identified among all the strains with overall similarity above 95%, with minor differences in the sequence similarity. Using the virulent gene data obtained from the Virulence Factor Database, 75 to 85 virulent genes were identified in the five genome assemblies with various key genes such as cytolethal distending toxin (cdt), type IV secretion system, cag PAI, plasticity region, cell-motility- and flagellar-associated genes, neutrophil-activating protein (HP-NAP), T4SS effector cytotoxin-associated gene A (cagA), and urease-associated genes ureA and ureB, etc. Sequence similarity between the virulence factors found in this study and reference genes was at least 90%. In summary, the results of our study showed the relationship between clinical results and specific H. pylori strains' (HPA1 and HPA2) genetics such as antibiotic resistance and specific virulence factors. These findings provide valued understanding of the epidemiology of H. pylori-associated diseases. Moreover, identification and genomics analysis have provided insights into the epidemiology, genetic diversity, pathogenicity, and potential drug resistance genes of H. pylori strains, offering a foundation for developing more targeted and effective medical interventions, including anti-virulent medications.

Unraveling the Radioprotective Mechanisms of UV-Resistant Bacillus subtilis ASM-1 Extracted Compounds through Molecular Docking.

Radioresistant microorganisms possess inimitable capabilities enabling them to thrive under extreme radiation. However, the existence of radiosensitive microorganisms inhabiting such an inhospitable environment is still a mystery. The current study examines the potential of radioresistant microorganisms to protect radiosensitive microorganisms in harsh environments. Bacillus subtilis strain ASM-1 was isolated from the Thal desert in Pakistan and evaluated for antioxidative and radioprotective potential after being exposed to UV radiation. The strain exhibited 54.91% survivability under UVB radiation (5.424 × 103 J/m2 for 8 min) and 50.94% to mitomycin-C (4 µg/mL). Extracellular fractions collected from ASM-1 extracts showed significant antioxidant potential, and chemical profiling revealed a pool of bioactive compounds, including pyrrolopyrazines, amides, alcoholics, and phenolics. The E-2 fraction showed the maximum antioxidant potential via DPPH assay (75%), and H2O2 scavenging assay (68%). A combination of ASM-1 supernatant with E-2 fraction (50 µL in a ratio of 2:1) provided substantial protection to radiosensitive cell types, Bacillus altitudinis ASM-9 (MT722073) and E. coli (ATCC 10536), under UVB radiation. Docking studies reveal that the compound supported by literature against the target proteins have strong binding affinities which further inferred its medical uses in health care treatment. This is followed by molecular dynamic simulations where it was observed among trajectories that there were no significant changes in major secondary structure elements, despite the presence of naturally flexible loops. This behavior can be interpreted as a strategy to enhance intermolecular conformational stability as the simulation progresses. Thus, our study concludes that Bacillus subtilis ASM-1 protects radiosensitive strains from radiation-induced injuries via biofilm formation and secretion of antioxidative and radioprotective compounds in the environment.

Genetic characterization of human echinococcosis in Southern Punjab, Pakistan.

Introduction: Echinococcosis is a neglected tropical zoonotic infection that affects both the human and livestock populations. In Pakistan, the infection is long-standing, but data on its molecular epidemiology and genotypic characterization in the southern Punjab region are limited. The aim of the current study was the molecular characterization of human echinococcosis in southern Punjab, Pakistan. Methods: Echinococcal cysts were obtained from a total of 28 surgically treated patients. Patients' demographic characteristics were also recorded. The cyst samples were subjected to further processing to isolate DNA in order to probe the Nad1 and Cyt-b genes, followed by DNA sequencing and phylogenetic analysis for genotypic identification. Results: The majority of the echinococcal cysts were from male patients (60.7%). The liver was the most commonly infected organ (60.71%), followed by the lungs (25%), spleen (7.14%), and the mesentery (7.14%). Molecular and genotypic identification through sequencing and phylogenetic tree analysis showed that most of the cysts (24/28, 85.7%) were caused by the species Echinococcus granulosus sensu stricto (E. granulosus s.s.) (G1 and G3), followed by Echinococcus multilocularis (E. multilocularis) and Echinococcus canadensis (E. canadensis) (G6/G7) (3/28, 10.8%, and 1/28, 3.5%, respectively). Conclusion: The current study concluded that the majority of human infections were caused by E. granulosus s.s., followed by the E. multilocularis and E. canadensis species (G6/G7). Genotypic characterization among both human and livestock populations is needed to explore the genetic diversity of echinococcosis.

Biological Evaluation and Computational Studies of Methoxy-flavones from Newly Isolated Radioresistant Micromonospora aurantiaca Strain TMC-15.

This study aims to determine UV-B resistance and to investigate computational analysis and antioxidant potential of methoxy-flavones of Micromonospora aurantiaca TMC-15 isolated from Thal Desert, Pakistan. The cellular extract was purified through solid-phase extraction and UV-Vis spectrum analysis indicated absorption peaks at λmax 250 nm, 343 nm, and 380 nm that revealed the presence of methoxy-flavones named eupatilin and 5-hydroxyauranetin. The flavones were evaluated for their antioxidant as well as protein and lipid peroxidation inhibition potential using di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium (DPPH), 2,4-dinitrophenyl hydrazine (DNPH), and thiobarbituric acid reactive substances (TBARS) assays, respectively. The methoxy-flavones were further studied for their docking affinity and interaction dynamics to determine their structural and energetic properties at the atomic level. The antioxidant potential, protein, and lipid oxidation inhibition and DNA damage preventive abilities were correlated as predicted by computational analysis. The eupatilin and 5-hydroxyauranetin binding potential to their targeted proteins 1N8Q and 1OG5 is - 4.1 and - 7.5 kcal/mol, respectively. Moreover, the eupatiline and 5-hydroxyauranetin complexes illustrate van der Waals contacts and strong hydrogen bonds to their respective enzymes target. Both in vitro studies and computational analysis results revealed that methoxy-flavones of Micromonospora aurantiaca TMC-15 can be used against radiation-mediated oxidative damages due to its kosmotrophic nature. The demonstration of good antioxidant activities not only protect DNA but also protein and lipid oxidation and therefore could be a good candidate in radioprotective drugs and as sunscreen due to its kosmotropic nature.

Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan.

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; blaAmpC 6 (100%), blaVIM-1, blaSHV and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.

Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes / Isolamento e triagem de bactérias resistentes ao cromo de resíduos industriais para fins de biorremediação

Abstract Chromium (VI) a highly toxic metal, a major constituent of industrial waste. It is continuously release in soil and water, causes environmental and health related issues, which is increasing public concern in developing countries like Pakistan. The basic aim of this study was isolation and screening of chromium resistant bacteria from industrial waste collected from Korangi and Lyari, Karachi (24˚52ʹ46.0ʺN 66˚59ʹ25.7ʺE and 24˚48ʹ37.5ʺN 67˚06ʹ52.6ʺE). Among total of 53 isolated strains, seven bacterial strains were selected through selective enrichment and identified on the basis of morphological and biochemical characteristics. These strains were designated as S11, S13, S17, S18, S30, S35 and S48, resistance was determined against varying concentrations of chromium (100-1500 mg/l). Two bacterial strains S35 and S48 showed maximum resistance to chromium (1600 mg/l). Bacterial strains S35 and S48 were identified through 16S rRNA sequence and showed 99% similarity to Bacillus paranthracis and Bacillus paramycoides. Furthermore, growth condition including temperature and pH were optimized for both bacterial strains, showed maximum growth at temperature 30ºC and at optimum pH 7.5 and 6.5 respectively. It is concluded that indigenous bacterial strains isolated from metal contaminated industrial effluent use their innate ability to transform toxic heavy metals to less or nontoxic form and can offer an effective tool for monitoring heavy metal contamination in the environment.

Resumo O cromo (VI), metal altamente tóxico, é um dos principais constituintes dos resíduos industriais. É liberado no solo e na água, causa problemas ambientais e de saúde de crescente preocupação pública em países em desenvolvimento como o Paquistão. O objetivo básico deste estudo foi o isolamento e a triagem de bactérias resistentes ao cromo de resíduos industriais coletados em Korangi e Lyari, Karachi (24˚52'46,0"N 66˚59'25,7"E e 24˚48'37,5"N 67˚06'52,6"E). Do total de 53 cepas isoladas, sete cepas bacterianas foram selecionadas por enriquecimento seletivo e identificadas com base em características morfológicas e bioquímicas. Essas cepas foram designadas como S11, S13, S17, S18, S30, S35 e S48, apresentaram alta resistência aos metais contra concentrações variáveis (100-1500 mg / l) de cromo. Já as cepas S35 e S48 foram identificadas por meio da sequência 16S rRNA e apresentaram 99% de similaridade com Bacillus paranthracis e Bacillus paramycoides. Além disso, as condições de crescimento incluindo temperatura e pH foram otimizadas e ambas as cepas bacterianas apresentaram crescimento máximo na temperatura de 30 ºC, enquanto seu pH ótimo foi observado em 7,5 e 6,5, respectivamente. Conclui-se que o potencial de resistência dessas bactérias resistentes ao cromo pode ser efetivamente utilizado na remoção de cromo de efluentes industriais contaminados. Técnicas de base biológica usando bactérias ajudarão a fornecer métodos mais baratos e ecológicos de remoção, recuperação e desintoxicação de cromo.

Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes / Isolamento e triagem de bactérias resistentes ao cromo de resíduos industriais para fins de biorremediação

Chromium (VI) a highly toxic metal, a major constituent of industrial waste. It is continuously release in soil and water, causes environmental and health related issues, which is increasing public concern in developing countries like Pakistan. The basic aim of this study was isolation and screening of chromium resistant bacteria from industrial waste collected from Korangi and Lyari, Karachi (24˚52ʹ46.0ʺN 66˚59ʹ25.7ʺE and 24˚48ʹ37.5ʺN 67˚06ʹ52.6ʺE). Among total of 53 isolated strains, seven bacterial strains were selected through selective enrichment and identified on the basis of morphological and biochemical characteristics. These strains were designated as S11, S13, S17, S18, S30, S35 and S48, resistance was determined against varying concentrations of chromium (100-1500 mg/l). Two bacterial strains S35 and S48 showed maximum resistance to chromium (1600 mg/l). Bacterial strains S35 and S48 were identified through 16S rRNA sequence and showed 99% similarity to Bacillus paranthracis and Bacillus paramycoides. Furthermore, growth condition including temperature and pH were optimized for both bacterial strains, showed maximum growth at temperature 30ºC and at optimum pH 7.5 and 6.5 respectively. It is concluded that indigenous bacterial strains isolated from metal contaminated industrial effluent use their innate ability to transform toxic heavy metals to less or nontoxic form and can offer an effective tool for monitoring heavy metal contamination in the environment.

O cromo (VI), metal altamente tóxico, é um dos principais constituintes dos resíduos industriais. É liberado no solo e na água, causa problemas ambientais e de saúde de crescente preocupação pública em países em desenvolvimento como o Paquistão. O objetivo básico deste estudo foi o isolamento e a triagem de bactérias resistentes ao cromo de resíduos industriais coletados em Korangi e Lyari, Karachi (24˚52’46,0”N 66˚59’25,7”E e 24˚48’37,5”N 67˚06’52,6”E). Do total de 53 cepas isoladas, sete cepas bacterianas foram selecionadas por enriquecimento seletivo e identificadas com base em características morfológicas e bioquímicas. Essas cepas foram designadas como S11, S13, S17, S18, S30, S35 e S48, apresentaram alta resistência aos metais contra concentrações variáveis (100-1500 mg / l) de cromo. Já as cepas S35 e S48 foram identificadas por meio da sequência 16S rRNA e apresentaram 99% de similaridade com Bacillus paranthracis e Bacillus paramycoides. Além disso, as condições de crescimento incluindo temperatura e pH foram otimizadas e ambas as cepas bacterianas apresentaram crescimento máximo na temperatura de 30ºC, enquanto seu pH ótimo foi observado em 7,5 e 6,5, respectivamente. Conclui-se que o potencial de resistência dessas bactérias resistentes ao cromo pode ser efetivamente utilizado na remoção de cromo de efluentes industriais contaminados. Técnicas de base biológica usando bactérias ajudarão a fornecer métodos mais baratos e ecológicos de remoção, recuperação e desintoxicação de cromo.

Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes

Abstract Chromium (VI) a highly toxic metal, a major constituent of industrial waste. It is continuously release in soil and water, causes environmental and health related issues, which is increasing public concern in developing countries like Pakistan. The basic aim of this study was isolation and screening of chromium resistant bacteria from industrial waste collected from Korangi and Lyari, Karachi (245246.0N 665925.7E and 244837.5N 670652.6E). Among total of 53 isolated strains, seven bacterial strains were selected through selective enrichment and identified on the basis of morphological and biochemical characteristics. These strains were designated as S11, S13, S17, S18, S30, S35 and S48, resistance was determined against varying concentrations of chromium (100-1500 mg/l). Two bacterial strains S35 and S48 showed maximum resistance to chromium (1600 mg/l). Bacterial strains S35 and S48 were identified through 16S rRNA sequence and showed 99% similarity to Bacillus paranthracis and Bacillus paramycoides. Furthermore, growth condition including temperature and pH were optimized for both bacterial strains, showed maximum growth at temperature 30ºC and at optimum pH 7.5 and 6.5 respectively. It is concluded that indigenous bacterial strains isolated from metal contaminated industrial effluent use their innate ability to transform toxic heavy metals to less or nontoxic form and can offer an effective tool for monitoring heavy metal contamination in the environment.

Resumo O cromo (VI), metal altamente tóxico, é um dos principais constituintes dos resíduos industriais. É liberado no solo e na água, causa problemas ambientais e de saúde de crescente preocupação pública em países em desenvolvimento como o Paquistão. O objetivo básico deste estudo foi o isolamento e a triagem de bactérias resistentes ao cromo de resíduos industriais coletados em Korangi e Lyari, Karachi (245246,0N 665925,7E e 244837,5N 670652,6E). Do total de 53 cepas isoladas, sete cepas bacterianas foram selecionadas por enriquecimento seletivo e identificadas com base em características morfológicas e bioquímicas. Essas cepas foram designadas como S11, S13, S17, S18, S30, S35 e S48, apresentaram alta resistência aos metais contra concentrações variáveis (100-1500 mg / l) de cromo. Já as cepas S35 e S48 foram identificadas por meio da sequência 16S rRNA e apresentaram 99% de similaridade com Bacillus paranthracis e Bacillus paramycoides. Além disso, as condições de crescimento incluindo temperatura e pH foram otimizadas e ambas as cepas bacterianas apresentaram crescimento máximo na temperatura de 30 ºC, enquanto seu pH ótimo foi observado em 7,5 e 6,5, respectivamente. Conclui-se que o potencial de resistência dessas bactérias resistentes ao cromo pode ser efetivamente utilizado na remoção de cromo de efluentes industriais contaminados. Técnicas de base biológica usando bactérias ajudarão a fornecer métodos mais baratos e ecológicos de remoção, recuperação e desintoxicação de cromo.

Epidemiology of Human Adenovirus in Pakistani Children Hospitalized with Community-Acquired Gastroenteritis under the Age of Five Years.

Acute gastroenteritis is the major cause of morbidity and mortality among infants and children around the globe. Along with other enteropathogens, human adenovirus (HadV) is a major etiological agent associated with diarrhea in young children. However, information about the epidemiology of Adenoviruses in Pakistan is limited or has not been reported. A total of 1082 stool samples were collected from patients with acute gastroenteritis under the age of five years with symptoms of diarrhea, vomiting, nausea, and abdominal cramps who visited Benazir Bhutto Hospital Rawalpindi and Children's hospital in Lahore of Punjab Province in Pakistan. Of this, 384 cases with no blood in their stool, negative for Rotavirus, and under the age of five years were recruited in this study. Human Adenoviruses were isolated in the human epithelial HEp-2 cell line. Furthermore, adenovirus antigen detection was carried out by an enzyme-linked immunosorbent assay (ELISA), and then all positive and negative samples were confirmed by nested PCR. After inoculating a clear stool supernatant on HEp-2 cell lines, we observed a positive cytopathic effect in 65 (16%) cases. Using an enzyme-linked immunosorbent assay, HAdV antigens were detected in 54 (14.06%) of the clear supernatant from gastroenteritis cases. However, HAdV hexon coding regions were amplified in 57 (14.80%) fecal samples, mainly from patients ≤24 months of age. The findings of this study suggest that adenovirus circulates significantly in the children population under the age of five years and may be the potential etiological factor of acute gastroenteritis in the mentioned cities. This study provides baseline data about the possible role of adenovirus in causing viral diarrhea in children. Further large-scale epidemiological surveys are recommended to better understand disease burden, etiological agents, and its clinical impact across the country.

Prebiotic potential of enzymatically prepared resistant starch in reshaping gut microbiota and their respond to body physiology.

The increase in consumer demand for high-quality food products has led to growth in the use of new technologies and ingredients. Resistant starch (RS) is a recently recognised source of fibre and has received much attention for its potential health benefits and functional properties. However, knowledge about the fate of RS in modulating complex intestinal communities, the microbial members involved in its degradation, enhancement of microbial metabolites, and its functional role in body physiology is still limited. For this purpose, the current study was designed to ratify the physiological and functional health benefits of enzymatically prepared resistant starch (EM-RSIII) from maize flour. To approve the beneficial health effects as prebiotic, EM-RSIII was supplemented in rat diets. After 21 days of the experiment, EM-RSIII fed rats showed a significant reduction in body weight gain, fecal pH, glycemic response, serum lipid profile, insulin level and reshaping gut microbiota, and enhancing short-chain fatty acid compared to control. The count of butyrate-producing and starch utilizing bacteria, such as Lactobacillus, Enterococcus, and Pediococcus genus in rat's gut, elevated after the consumption of medium and high doses of EM-RSIII, while the E. coli completely suppressed in high EM-RSIII fed rats. Short-chain fatty acids precisely increased in feces of EM-RSIII feed rats. Correlation analysis demonstrated that the effect of butyrate on functional and physiological alteration on the body had been investigated during the current study. Conclusively, the present study demonstrated the unprecedented effect of utilising EM-RSIII as a diet on body physiology and redesigning gut microorganisms.

Microbial Pretreatment of Chicken Feather and Its Co-digestion With Rice Husk and Green Grocery Waste for Enhanced Biogas Production.

To utilize wastes and residues sustainably and excellently, there is a need to fend for efficient methods and resources for biogas production. Use of poultry waste for biogas production represents one of the most important routes toward reaching global renewable energy targets. The current study involves microbial pretreatment of chicken feather waste, followed by its co-digestion with rice husk and green grocery waste in batch and continuous reactors, respectively. Microbial pretreatment of chicken feathers by keratinase secreting Pseudomonas aeruginosa was an effective and eco-friendly approach to make its recalcitrant structure available as a raw substrate for biogas production. The current study also addressed the enhancement and stability of anaerobic digestion by co-digestion. Results demonstrated that biogas production was increased by microbial pretreatment of chicken feathers and that the percentage increase in biogas yield was 1.1% in microbialy pretreated feathers compared to mono-digestion (non-pretreated feathers) in batch fermentation. The highest yield of biogas was obtained in a batch reactor having co-digestion of pretreated rice husk and microbial pretreated chicken feathers. The co-digestion of chicken feathers hydrolysate with green grocery waste in continuous fermentation mode has also enhanced the biogas yield as compared to average of mono-digestion (chicken feather hydrolysate and green grocery waste) and, therefore, improve the efficiency of the overall process.

Isolation and molecular characterization of Adenovirus in suspected acute flaccid paralysis patients: A preliminary report from Pakistan.

Human adenoviruses (HAdVs) usually cause asymptomatic or mild infection, but infrequently, they are responsible for various severe syndromes including neurological disorders. Various research studies have investigated the association of HAdVs with acute flaccid paralysis (AFP). The purpose of this study was to investigate the genetic diversity of HAdVs and their association with AFP. Stool samples from patients ≤ 12 years of age with suspected AFP were collected from all over Pakistan within the framework of poliovirus surveillance. Poliovirus- and enterovirus-negative samples were screened for HAdVs. For virus isolation, the human epithelial cell line HEp-2c was used, culture-positive samples were screened by nested PCR assay, and partial hexon gene sequences were used for genotype identification. Out of 172 samples, 94 were positive by virus isolation, 89 were positive by PCR, and 32 isolates were genotyped successfully. Phylogenetic analysis showed that the HAdVs belonged to species A (HAdV-A12 and A31), B (HAdV-B3 and B7), C (HAdV-C1 and C6), D (HAdV-D19 and D93), and F (HAdV-F41), showing 99-100% nucleotide sequence identity and 98.3-100% amino acid sequence identity). Most of these genotypes have been reported previously in AFP cases, but this is the first report of the detection of HAdV-D93 in stool samples from AFP cases. The detection of a significant fraction of the HAdVs genotypes indicates that these genetically distinct genotypes are circulating in Pakistan and suggests their possible role in the pathogenesis of AFP.

Positivity, diagnosis and treatment follow-up of cutaneous leishmaniasis in war-affected areas of Bajaur, Pakistan.

This study was conducted to explore the frequency of positivity of cutaneous leishmaniasis (CL) in the tribal district Bajaur located near the Pak-Afghan border. The present study was conducted at the Leishmaniasis Center of Headquarter Hospital Khar District Bajaur, Pakistan. In total, 646 patients were recruited and included in the study after ethical approval and consent from the patients. CL was confirmed by taking blood samples from the sides of the lesion and observing them under a microscope using Giemsa staining. Information about demographic factors was collected from the study participants with a questionnaire and analyzed by SPSS. It was found that 73.8% of suspected patients were positive and 26.2% were negative for CL. There were 51.9% male and 48.1% female patients. The most frequently affected site was the face (42.6%), and most of the patients (85.8%) had only one lesion. The positivity of CL was higher among those under age 15 years. The area of most positivity, with 45.2% of the cases, was Tehsil Mamund. Most of the patients (46.6%) lived in stone houses, with 98.6% of patients having domestic animals in their houses. Approximately 198 patients were treated with intramuscular and intralesional injections of meglumine antimoniate, and their weekly follow-up revealed that 48% of patients recovered, while the remaining patients left the course of treatment at different stages of therapy. The positivity of CL is high in this area and is confirmed by the detection of Leishmania amastigotes in the blood collected from their lesions. Socioeconomic factors are the main underlying causes of the rapid spread of this disease and meglumine antimoniate is an effective drug.

Degradation of lignin by Bacillus altitudinis SL7 isolated from pulp and paper mill effluent.

Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40 °C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.

Production of free fatty acids from various carbon sources by Ogataea polymorpha.

Energy shortage and environmental concern urgently require establishing the feasible bio-refinery process from various feedstocks. The methylotrophic yeast Ogataea polymorpha is thermo-tolerant and can utilize various carbon sources, such as glucose, xylose and methanol, which makes it a promising host for bio-manufacturing. Here, we explored the capacity of O. polymorpha for overproduction of free fatty acids (FFAs) from multiple substrates. The engineered yeast produced 674 mg/L FFA from 20 g/L glucose in shake flask and could sequentially utilize the mixture of glucose and xylose. However, the FFA producing strain failed to survive in sole methanol and supplementing co-substrate xylose promoted methanol metabolism. A synergistic utilization of xylose and methanol was observed in the FFA producing strain. Finally, a mixture of glucose, xylose and methanol was evaluated for FFA production (1.2 g/L). This study showed that O. polymorpha is an ideal host for chemical production from various carbon sources.

Cloning, biochemical characterization and molecular docking of novel thermostable ß-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin.

This study reports a novel BglA9 gene of 1345 bp encoding ß-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 µmol/min/mg), kcat (38.43 s-1) and kcat/Km (135.5 s-1 mM-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 µmol/min/mg, 18.28 s-1and 3.27 s-1 mM-1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

Prevalence and Molecular Characterization of Cystic Echinococcosis in Livestock Population of the Malakand Division, Khyber Pakhtunkhwa, Pakistan.

Cystic echinococcosis (CE) is a neglected zoonotic disease prevalent in Pakistan, but the genetic diversity of the cestode is largely unexplored in the country. This study investigated the molecular epidemiology of CE infecting the livestock population of the Malakand division, Khyber Pakhtunkhwa, Pakistan. A total of 1,200 livestock, including buffaloes, cattle, goats, and sheep, were examined for echinococcosis from November 2017-2018 at different slaughterhouses in the Malakand division. Hydatid cysts were collected from different organs, and hydatid cyst fluid (HCF) was examined microscopically and used for DNA extraction. The LSU (rrnl) and NAD1 genes were amplified and sequenced. The overall prevalence of CE was 17% (204/1,200), including cows (21.7%), buffaloes (17.4%), goats (10%), and sheep (9.6%). The infection was relatively more prevalent among males (17%) than females (16.9%) and animals of older age (>5 years) (p = 0.710). Liver (63.2%) and lungs (25%) were more affected as compared to kidneys (6.8%) and heart (4.9%). HCF analysis indicated that 52.0% of the cysts were sterile and (48.0%) were fertile. Sequencing and phylogenetic analyses confirmed 80.0% of the isolates as Echinococcus granulosus sensu stricto (G1-G3) in all animal species, while Echinococcus equinus (G4) and Echinococcus ortleppi (G5) were present in buffaloes. The present study concluded that CE is prevalent in the livestock population of Malakand. Besides E. granulosus s. s. (G1-G3), E. ortleppi genotype (G5) and E. equinus (G4) in livestock were also reported.