Molecular basis for the increased activity of ZMS-2 serine protease in the presence of metal ions and hydrogen peroxide.

Serine proteases are important enzymes widely used in commercial products and industry. Recently, we identified a new serine protease from the desert bacterium Bacillus subtilis ZMS-2 that showed enhanced activity in the presence of Zn2+, Ag+, or H2O2. However, the molecular basis underlying this interesting property is unknown. Here, we report comparative studies between the ZMS-2 protease and its homolog, subtilisin E (SubE), from B. subtilis ATCC 6051. In the absence of Zn2+, Ag+, or H2O2, both enzymes showed the same level of proteolytic activity, but in the presence of Zn2+, Ag+, or H2O2, ZMS-2 displayed increased activity by 22%, 8%, and 14%, whereas SubE showed decreased activity by 16%, 12%, and 9%, respectively. In silico studies showed that both proteins have almost identical amino acid sequences and folding structures, except for two amino acids located in the protruding loops of the proteins. ZMS-2 contains Ser236 and Ser268, whereas SubE contains Thr236 and Thr268. Replacing Ser236 or Ser268 in ZMS-2 with threonine resulted in variants whose activities were not enhanced by Zn2+ or Ag+. However, this single mutation did not affect the enhancement by H2O2. This finding may be used as a basis for engineering better proteases for industrial uses.

Characterization and Pilot-Scale Application of the ZMS-2 Serine Protease with Novel Properties for the Eco-friendly Leather Processing.

Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg-1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn+2 (204%), Ag+ (110%), H2O2 (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL-1), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

In-vitro evaluation of virulence markers and antifungal resistance of clinical Candida albicans strains isolated from Karachi, Pakistan.

Candidiasis is a significant fungal infection with high mortality and morbidity rates worldwide. Candida albicans is the most dominant species responsible for causing different manifestations of candidiasis. Certain virulence traits as well as its resistance to antifungal drugs contribute to the pathogenesis of this yeast. This study was designed to determine the production of some virulence factors, such as biofilm formation and extracellular hydrolytic enzymes (esterase, coagulase, gelatinase, and catalase) by this fungus, as well as its antifungal resistance profile. A total of 304 clinical C. albicans isolates obtained from different clinical specimens were identified by a conventional diagnostic protocol. The antifungal susceptibility of C. albicans strains was determined by disk diffusion technique against commercially available antifungal disks, such as nystatin 50 µg, amphotericin B 100 unit, fluconazole 25 µg, itraconazole 10 µg, ketoconazole 10 µg, and voriconazole 1 µg. The assessment of biofilm formation was determined by the tube staining assay and spectrophotometry. Gelatinase, coagulase, catalase, and esterase enzyme production was also detected using standard techniques. A total of 66.1% (201/304) and 28.9% (88/304) of C. albicans strains were susceptible-dose dependent (SDD) to nystatin and itraconazole, respectively. Among the antifungal drugs, C. albicans strains showed high resistance to ketoconazole 24.7% (75/304); however, no statistically significant relationship between the clinical origin of C. albicans isolates and antifungal drug resistance pattern was detected. For virulence factors, the majority of the C. albicans strains actively produced biofilm and all hydrolytic enzymes. Biofilm formation was demonstrated by 88% (267/304) of the strains with a quantitative mean value 0.1762 (SD ± 0.08293). However, 100% (304/304) of isolates produced catalase enzyme, 69% (211/304) produced coagulase, 66% (197/304) produced gelatinase, and 52% (157/304) produced esterase enzyme. A significant relationship between the source of specimens and biofilm formation by C. albicans was observed; nevertheless, there was no significant relationship between different sources of C. albicans strains and the production of different enzymatic virulence factors. The study found that C. albicans strains have excellent potential to produce virulence markers and resistance to antifungals, which necessitates surveillance of these opportunistic pathogens to minimize the chances of severe invasive infections.

Characterization of the genome and serine protease of a novel Bacillus subtilis isolate.

Current study was undertaken to carry out the genome-wide analysis of a multipotent isolate from desert soil which was previously identified as Bacillus tequilensis based on 16S rDNA analysis. This study also aims to characterize the serine protease and its biocatalytic potentials implying a combination of empirical and in-silico approaches. Next generation sequencing and short read de novo assembly generated the 4,235,084 bp draft genome of Bacillus sp. ZMS-2. Genome sequence analysis by digital DNA:DNA hybridization (dDDH) and average nucleotide identity classified the isolate as Bacillus subtilis ZMS-2 (Bioproject ID: PRJNA691551). Genome annotation revealed 10 antibiotic resistance genes, 8 antibiotic/antifungal gene clusters and 25 genes encoding proteases including subtilisin E, an extracellular alkaline protease. This extracellular protease (ZMS-2 protease) was produced using a statistically optimized medium, purified partially and characterized as alkaline serine protease. The partially purified ZMS-2 protease (780 U/mL) showed a 21 mm zone of casein hydrolysis and dehaired goat skin by pulling out hair with roots. These catalytic potentials of ZMS-2 protease were further confirmed using scanning electron microscopy of casein beads and dehaired skin. The study concludes B. subtilis ZMS-2 as a potent producer of a protease with promising potentials of commercial importance.

In vivo hepatoprotective and in vitro antimicrobial potential of Ceasalpinia bonduc (Linn): Pharmacological correlation with identified phytochemicals.

The in vivo hepatoprotective potential of methanolic extract of Ceasalpinia bonduc (CBLM) has been explored against carbon tetrachloride (CCl4) induced acute liver injury in rats. Treatment of plant extract on CCl4 intoxicated liver significantly reduced the hepatoxicity, along with serum enzymes GPT and GOT. To explore the chemical constituents from CBLM extract, it was fractionated into non-polar to moderately polar fractions (CBLM-H, CBLM-HEt, CBLM-Et, CBLM-EtM, CBLM-M) and subjected to GC/GC-MS analysis. Altogether twenty seven (~71%) phytochemicals were identified from different fractions by using Electronic Mass Spectral Library GC-MS (NIST 20). Out of which twenty one are first time reported from Ceasalpinia bonduc, fourteen from genus Caesalpinia and ten from family Fabaceae. The identified phytochemicals 2-ethyl-2-hydroxy-1,3-dimethylcyclopentanecarboxylic acid, ethyl ester (21) and 1,3,5-triazine-2,4-diamine,6-hydroxy-N,N-dicyclohexyl (23) are first time identified as plant metabolites. To explore the antimicrobial potential four strains of Gram-positive and eight strains of Gram-negative bacteria were used along with pure cultures of five saprophytic fungus (molds) and two strains of yeast were utilized. CBLM-H and CBLM-HEt were exhibited praiseworthy antimicrobial potential. CBLM-H showed complete growth inhibition of P. mirabilis and V. cholerae at the concentration of 0.1g/mL while CBLM-HEt at 0.05g/mL halted the growth of S. aureus.

Luffa cylindrica Immobilized with Aspergillus terreus QMS-1: an Efficient and Cost-Effective Strategy for the Removal of Congo Red using Stirred Tank Reactor.

Microbial populations within the rhizosphere have been considered as prosperous repositories with respect to bioremediation aptitude. Among various environmental contaminants, effluent from textile industries holds a huge amount of noxious colored materials having high chemical oxygen demand concentrations causing ecological disturbances. The study was aimed to explore the promising mycobiome of rhizospheric soil for the degradation of azo dyes to develop an efficient system for the exclusion of toxic recalcitrants. An effluent sample from the textile industry and soil samples from the rhizospheric region of Musa acuminata and Azadirachta indica were screened for indigenous fungi to decolorize Congo red, a carcinogenic diazo dye, particularly known for its health hazards to the community. To develop a bio-treatment process, Aspergillus terreus QMS-1 was immobilized on pieces of Luffa cylindrica and exploited in stirred tank bioreactor under aerobic and optimized environment. Quantitative estimation of Congo red decolorization was carried out using UV-Visible spectrophotometer. The effects of fungal immobilization and biosorption on the native structure of Luffa cylindrica were evaluated using a scanning electron microscope. A. terreus QMS-1 can remove (92%) of the dye at 100 ppm within 24 h in the presence of 1% glucose and 1% ammonium sulphate at pH 5.0. The operation of the bioreactor in a continuous flow for 12 h with 100 ppm of Congo red dye in simulated textile effluent resulted in 97% decolorization. The stirred tank bioreactor was found to be a dynamic, well maintained, no sludge producing approach for the treatment of textile effluents by A. terreus QMS-1 of the significant potential for decolorization of Congo red.Microbial populations within the rhizosphere have been considered as prosperous repositories with respect to bioremediation aptitude. Among various environmental contaminants, effluent from textile industries holds a huge amount of noxious colored materials having high chemical oxygen demand concentrations causing ecological disturbances. The study was aimed to explore the promising mycobiome of rhizospheric soil for the degradation of azo dyes to develop an efficient system for the exclusion of toxic recalcitrants. An effluent sample from the textile industry and soil samples from the rhizospheric region of Musa acuminata and Azadirachta indica were screened for indigenous fungi to decolorize Congo red, a carcinogenic diazo dye, particularly known for its health hazards to the community. To develop a bio-treatment process, Aspergillus terreus QMS-1 was immobilized on pieces of Luffa cylindrica and exploited in stirred tank bioreactor under aerobic and optimized environment. Quantitative estimation of Congo red decolorization was carried out using UV-Visible spectrophotometer. The effects of fungal immobilization and biosorption on the native structure of Luffa cylindrica were evaluated using a scanning electron microscope. A. terreus QMS-1 can remove (92%) of the dye at 100 ppm within 24 h in the presence of 1% glucose and 1% ammonium sulphate at pH 5.0. The operation of the bioreactor in a continuous flow for 12 h with 100 ppm of Congo red dye in simulated textile effluent resulted in 97% decolorization. The stirred tank bioreactor was found to be a dynamic, well maintained, no sludge producing approach for the treatment of textile effluents by A. terreus QMS-1 of the significant potential for decolorization of Congo red.

Prevalence and association of HBV and HCV infection with cardiovascular disease risk factors in a peri-urban population.

OBJECTIVE: To investigate the possible associations of hepatitis B and C virus infection with cardiovascular disease risk factors inperi-urban population. METHODS: The cross-sectional study was conducted from February to December 2016 in the periurban low-resource locality of Bin Qasim Town in Karachi. Serum samples were screened for hepatitis B surface antigen and hepatitis C virus antibodies. Anthropometric measurements were taken and markers related to cardiovascular disease were examined. Association of the two hepatitis virus infections with cardiovascular diseasewere investigated by anaylsing the data using SPSS 16. RESULTS: There were 691 subjects. Serum triglyceride levels were significantly low in patients with hepatitis B virus (p<0.05). Those with hepatitis C virus had markedly low total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglyceride levels (p<0.05 each), whereas random blood sugar and body mass index values were significantly high (p<0.05 each. Hepatitis C virus infection was positively associated with body mass index and random blood glucose, and inversely associated with total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol(p< 0.05 each). CONCLUSIONS: Hepatitis B virus infection showed a significant inverse association with triglyceride levels. However, hepatitis C virus infection was positively associated with body mass index and random blood sugar, and inversely associated with total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels.

Bacillus tequilensis ZMS-2: A novel source of alkaline protease with antimicrobial, anti-coagulant, fibrinolytic and dehairing potentials.

Emerging resistance to existing antimicrobial agents is one of the growing concerns and a serious problem for public health globally. Currently available antimicrobial agents are potent and effective but surfacing resistance to these drugs has not been ruled out so far. Therefore, it is utmost important to explore new bioactive compounds from natural sources to meet future needs. The present study was designed to produce, optimize, characterize and evaluate antimicrobial, fibrinolytic and anti-coagulant potential of a new alkaline protease. Proteolytic strain from desert soils of Tharparkar, Pakistan was subjected to 16S rDNA sequencing and identified as Bacillus tequilensis ZMS-2(Genbank Accession No. MK101013). During submerged fermentation at 37ºC, maximum enzyme production (454 U/ml) was observed with 24h old inoculum. The best incubation time was 72h (544 U/ml), optimum inoculum size and pH was 10% at pH 8 with 494 and 506 U/ml, respectively. The best carbon source was starch (571 U/ml), while ideal substrate was wheat bran (536 U/ml). Optimal temperature and pH for proteolytic activity was 60ºC (420 U/ml) and 8 (332 U/ml). Alkaline protease showed antibacterial activity against Staphylococcus aureus (27mm), Bacillus licheniformis (20mm), Klebsiella pneumoniae (17mm) and Escherichia coli (15mm). The strain B. tequilensis ZMS-2 also exhibited anticoagulant, fibrinolytic and dehairing potential suggesting application of its protease in various industries.

Non-albicans Candida species: Emergence of neglected pathogens among population of Karachi.

Candida albicans was considered as the principal cause of opportunistic candidiasis but nowadays, neglected non-albicans Candida (NAC) species are evolving as more virulent and drug resistant strains. This research was intended to assess pervasiveness of candidiasis mainly caused by NAC species in Karachi city. A total of 562 clinical isolates of Candida spp. collected during the period of one year were identified by microscopic as well as morphological (germ tube formation, characteristics on CHROM agar and Corn meal agar) and Biochemical (sugar assimilation and fermentation) characteristics. Doubtful species were further identified by using Remel RapIDTM yeast plus kit. The results were statistically analyzed by SPSS 16.0 version software. Isolated strains of candida revealed slight predominance of C. albicans (54.5%) over non- albicans Candida species (45.5%). Among NAC species, C. tropicalis and C. glabrata were isolated as the predominant species. These clinical species were procured mainly from urine samples of females (73.7%) of age group 20-30 years. No significant correlations exist between Candida species and their months of isolation as well as their isolation from different districts of Karachi. Emergence of NAC species may predict an upcoming threat in health care facilities and hence, require prompt management and accurate identification to suggest empirical antifungal therapy.

Antimicrobial potential of newly isolated Aspergillusterreus MK-1: An approach towards new antibiotics.

OBJECTIVE: To attempt discovering new bioactive metabolites from fungal sources. METHODS: The exploratory study was conducted at the Department of Microbiology, Federal Urdu University for Arts, Science and Technology, Karachi from January 2016 to November 2017and comprised of soil samples collected from rhizosphere region of different garden plants from the city. Fungi were screened for production of antibiotics by testing cell-free culture filtrates obtained by Shake-flask fermentation technique. Agar-Well diffusion assay method was used to evaluate antagonistic activity against pathogenic microorganisms. RESULTS: Bioactive compounds extracted by ethyl acetate and thin layer chromatography revealed mixture of compounds in the crude extract. AspergillusterreusMK-1 showed significant inhibition of medically important test pathogens namely Staphylococcus aureus, Pseudomonas aeruginosa, Escherichiacoli, Salmonella typhi, Micrococcus luteus, Streptococcus epidermidis, Bacillus subtilis, Candida albicans and Aspergillusniger. The best biological activity of crude ethyl acetate extract was observed against Pseudomonas aeruginosa (63mm). CONCLUSIONS: Newly isolated AspergillusterreusMK-1 emerged as a potent candidate for the production of antimicrobial compounds.

Fungal contamination in smokeless tobacco products traditionally consumed in Pakistan.

OBJECTIVE: To isolate potential pathogenic fungi from smokeless tobacco products. METHODS: The study was conducted from January 2015 to February 2017 during which samples of smokeless tobacco products such as Mainpuri, Tambako, Khiwam, Gutkha, Naswar and Mawa etc. were collected from different cities of Pakistan. The samples were tested for fungal contamination by spread plate method. Different strains of fungi were isolated and identified on the basis of their macroscopic as well as microscopic characteristics. The fungal strains isolated were also screened for their susceptibility to commonly used antifungal drugs by disc diffusion method. RESULTS: Of the 600 samples collected, 300(50%) were from Sindh, 70(11.7%) Balochistan, 74(12.3%) from Khyber Pakhtunkhwa, 105(17.5%) from Punjab and 51(8.5%) from Azad Kashmir. In terms of products, there were 404(67.3%) samples of Naswar, 69(11.5%) Patti, 40(6.6%) Khiwam, 35(5.8%) Mawa, 32(5.3%) Gutkha, and 20(3.3%) Mainpuri samples. Different species of Aspergillus were predominantly isolated followed by Penicillium, Mucor, Sepedonium and Trichophyton. The isolated strains of Aspergillus also revealed resistance against many commonly-used anti-fungals such as Amphotericin B and Itraconazole.. CONCLUSIONS: There was high prevalence of opportunistic fungi in study samples, posing a threat for human health which requires prompt notice and management.

Isolation of potentially pathogenic fungi from selected pigeons' feeding sites in Karachi: A new dimension to health hazard.

OBJECTIVE: To determine the presence of pathogenic fungal strains in areas where pigeons are present in a large number. METHODS: This study was conducted at the Federal Urdu University of Arts, Science and Technology, Karachi, from February 2015 to March2016, and comprised samples of soil contaminated with pigeons' excreta. The samples were collected from 20 different pigeon-feeding places in the city. These samples were processed for the isolation and identification of fungi by using standard conventional methods. The fungal strains isolated were also tested for their susceptibility to commonly used antifungal agents by disc diffusion technique. RESULTS: There were 105 samples. A wide variety of fungal strains belonging to different genera of Aspergillus, Rhizopus, Penicillium, Fusarium and Candida were isolated and identified by using conventional methods. The antifungal resistance pattern of these strains also depicts emergence of resistance against commonly used antifungal agents such as amphotericin B and fluconazole. CONCLUSIONS: The soil and air of places densely populated with pigeons were found to be loaded with fungal spores and many of them were potential pathogens.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189.

Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189

Abstract Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.

Multi-drug resistant pseudomonas aeruginosa: a threat of nosocomial infections in tertiary care hospitals.

OBJECTIVE: To determine the resistance patterns of Pseudomonas aeruginosa to currently available anti-pseudomonal drugs and frequency of nosocomial infections caused by multi drug resistant Pseudomonas aeruginosa in hospitals. METHODS: Clinical isolates of Pseudomonas aeruginosa were collected from patients admitted in different hospitals of Karachi between July 2012 and June 2013. The isolates were identified by conventional and Analytical Profile Index 20NE kit methods while the antibiograms of these isolates were determined by Kirby-Bauer disc diffusion method. RESULTS: Of the 204 isolates, 79(39%) were obtained from intensive care units: Overall, 135(66%) isolates belonged to men, and 35(17.2%) belonged to 10-15 year age group. The overall antibiogram pattern showed high resistance to commonly used antibiotics like Ofloxacin 125(61.3%), Cefepime 117(57.3%), Ceftazidime 110(53.9%), Amikacin 108(53%). Of all the isolates, 129(63.2%) were considered multidrug resistant. The most effective antibiotics were Colistin, Polymyxin B and Meropenem. CONCLUSION: Increasing multidrug resistance among nosocomial pathogens is an alarming situation in a hospital setting and requires prompt management of these cases.