Unveiling the genomic potential of a novel thermostable glycoside hydrolases producing Neobacillus sedimentimangrovi UE25.

Genetic and enzymatic potential of Neobacillus sedimentimangrovi has not been assembled to date. Here, we report a high-quality genome assembly of thermophilic bacterium Neobacillus sedimentimangrovi UE25 using Illumina Hi-seq 2500. The strain was isolated from a crocodile pond Manghopir, Karachi, Pakistan. QUAST quality parameters showed 37.75% GC content and exhibited the genome into 110 contigs, with a total size of 3,230,777 bases. Genome of N. sedimentimangrovi UE25 harbors phage mediated DNA through horizontal gene exchange from the phages, symbiotic and pathogenic bacteria. Most of the phage genome encodes for hypothetical proteins, protease, and phage assembly proteins. Gene clusters encoding the intrinsic resistance to glycopeptides, isoniazid, rifamycin, elfamycin, macrolide, aminoglycosides, tetracycline and fluoroquinolone were identified into the genome. Since, the strain has been reported for the production of many industrially important thermostable enzymes, therefore, the genomic data of thermostable enzymes might be helpful to employ this species in commercial sectors. Probing genes of multiple thermostable glycoside hydrolase enzymes especially xylanases of N. sedimentimangrovi UE25 showed genetic diversity among the genes and confer the industrial importance of this microorganism. Furthermore, the genome of N. sedimentimangrovi will greatly improve our understanding of its genetics and evolution.

Designing Functionally Substituted Pyridine-Carbohydrazides for Potent Antibacterial and Devouring Antifungal Effect on Multidrug Resistant (MDR) Strains.

The emergence of multidrug-resistant (MDR) pathogens and the gradual depletion of available antibiotics have exacerbated the need for novel antimicrobial agents with minimal toxicity. Herein, we report functionally substituted pyridine carbohydrazide with remarkable antimicrobial effect on multi-drug resistant strains. In the series, compound 6 had potent activity against four MDR strains of Candida spp., with minimum inhibitory concentration (MIC) values being in the range of 16-24 µg/mL and percentage inhibition up to 92.57%, which was exceptional when compared to broad-spectrum antifungal drug fluconazole (MIC = 20 µg/mL, 81.88% inhibition). Substitution of the octyl chain in 6 with a shorter butyl chain resulted in a significant anti-bacterial effect of 4 against Pseudomonas aeruginosa (ATCC 27853), the MIC value being 2-fold superior to the standard combination of ampicillin/cloxacillin. Time-kill kinetics assays were used to discern the efficacy and pharmacodynamics of the potent compounds. Further, hemolysis tests confirmed that both compounds had better safety profiles than the standard drugs. Besides, molecular docking simulations were used to further explore their mode of interaction with target proteins. Overall results suggest that these compounds have the potential to become promising antimicrobial drugs against MDR strains.

Thymol mitigates cadmium-induced behavioral and cognitive deficits by up-regulating hippocampal BDNF levels in rats.

Cadmium is a potent neurotoxin and induces adverse impact on brain function. Protective effects of monoterpenes on the CNS have been reported previously. The present study was designed to investigate the beneficial effect of thymol on cadmium-induced neurotoxicity. Rats were initially divided into 2 groups, vehicle control and thymol. Thymol (40mg/kg) was given orally for 14 days. Each group was subdivided into two groups (Vehicle control and Cadmium, Thymol and Thymol+Cadmium). Cadmium Chloride (5mg/kg) was given for last 3 days only to the groups assigned as Cadmium and Thymol+Cadmium. Behavioral parameters were assessed after 24h of last dose of cadmium. Brain sample were collected and BDNF was measured in hippocampus. The present study suggests that pre-administration of thymol provides a protective therapy against cadmium-induced intoxication by enhancing the brain BDNF levels and plasticity. Results further suggest that thymol not only ameliorates cadmium-induced learning and memory impairment but also reduced anxiety, motor incoordination and depression assessed by various behavioral tests. The study may provide a better apprehension of the neuroprotective role of thymol and highlighting its significance in the diet for human health particularly in cadmium intoxication.

Surviving under pollution stress: Antibacterial and antifungal activities of the Oyster species (Magallana bilineata and Magallana cuttackensis).

Antibiotic effectively controls the bacterial and fungal infections. Pathogens developing multi-drug resistance are a global health concern, which necessitate developing new molecules to overcome the resistance dilemma. This study explores the intrinsic ability of marine oysters synthesizing bioactive compounds. The tissue extracts prepared in n-hexane from two oysters, Magallana bilineata and Magallana cuttackensis compared for broad spectral antimicrobial activates against the fungal, Gram positive and negative pathogens. Regardless, both the species tolerated the same pollution indices; the M. bilineata exhibits stronger antimicrobial activities as compared to M. cuttackensis. M. bilineata potentially inhibited the bacterial growth with minimal inhibitory concentration (0.75-20 µg/ml) and fungal pathogens (0.75-5 µg/ml) as compared to ciprofloxacin and miconazole. Inhibitory potential complimented with reduce bactericidal and fungicidal concentrations required to observed susceptible zone of inhibition (ZOI). The inhibition augmented with increased antimicrobial index (AMI) and total activity index (TAI) against the human pathogen than those of M. cuttackensis. It is widely acknowledged that there is a need to develop novel antimicrobial agents to minimize the threat of emerging multiple antimicrobial resistant pathogens. Therefore, the oysters surviving in the pollution stress modulate the physiological and immune response may exploit to develop potential antibiotics.

Molecular assessment of virulence determinants, hospital associated marker (IS16gene) and prevalence of antibiotic resistance in soil borne Enterococcus species.

Enterococci, no more regarded as GRAS (Generally Recognized As Safe) organism, are emerging as an important source of nosocomial infections worldwide. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes. We aimed to examine the prevalence, dissemination, antibiotic resistance and virulent factors associated with enterococci from bulk soil (BS). A total of 372 enterococci were isolated from 500 soil samples. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E. faecium (77%), E. faecalis (10%), E. hirae (4%) and E. casseliflavus (1%) were the major species isolated. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E. faecalis isolates. The E. faecium carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E. faecium, while enlA (18%) and ef1097 (2.6%) was only detected in E. faecalis isolates. 50% E. faecalis and 2% E. faecium isolates harbored IS16, while five E. faecalis harbored both IS16 and espTIM genes providing strong evidence about the presence of espTIM gene on 64 Kb pathogenicity island. BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major antibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. Presence of multiple antibiotic resistant, virulent and hospital associated enterococci in bulk soil represents a potential source for further dissemination to humans and animals and poses potential impact on public health.

Prevalence and genetic profiling of tetracycline resistance (Tet-R) genes and transposable element (Tn916) in environmental Enterococcus species.

Resistance against antimicrobial agents in enterococci is a global concern that not only challenges infection therapy but also make them reservoir of antibiotic resistance in human and animal alike. This study was conducted to establish tetracycline resistance profiles, prevalence of tet genes and transposable element (Tn916) in enterococcal soil and clinical isolates. Enterococci (n = 1210) from different environmental niche were collected and subjected to molecular identification. In total, 361 isolates showed tetracycline resistance at the breakpoint of 32 µg ml-1. MICs (32-512 µg ml-1) were established by both agar and micro-broth dilution methods. Soil isolates (n = 76) were further investigated for Tet genes (tet-A, C, K, L, M, S, O) and Tn916. Major resistance was observed in E. faecium 67% followed by E. faecalis 22%, E. hirae 8% and E. casseliflavus 2.6%. Results revealed that tet(L) was more frequently found in E. faecium 74.5%, while tet(M) was in high prevalence in E. faecalis 82.3%. Tn916 was detected in both clinical and soil isolates (i.e. 43.3% and 19.7%, respectively). RAPD-PCR analysis showed high diversity among the investigated isolates. Cumulatively, our results revealed high-level tetracycline resistance and the presence of multiple Tet genes and transposable element Tn916 in enterococci.

Molecular Assessments of Antimicrobial Protein Enterocins and Quorum Sensing Genes and Their Role in Virulence of the Genus Enterococcus.

Enterococcus has emerged as an opportunistic pathogen because of its antibiotic resistance and virulence profile, which makes it a causative agent of several diseases like endocarditis, surgical site, and urinary tract infections. Currently, species of this genus are the 2nd most frequently isolated microorganisms from hospital-acquired infections. Significant association with hospitals and unhygienic conditions of the environments has made them resistant to a wide range of antibiotics. On the brighter side, enterococci have the ability to produce antimicrobial proteins (i.e., enterocins) that exhibit wide antagonistic activity, thus making them useful microbes in the food and pharmaceutical industries. Enterocins are also involved in niche control in gut microbiota which is regulated by the quorum sensing (QS) system. A bacterial communication system that is controlled by the fsr operon in enterococci consists of FsrABDC, ef1097, and GelE/SprE genes. Hence, the present study was conducted for molecular assessment of enterocins and quorum sensing genes, inter-environmental correlation, and species prevalence of enterococci isolated from different environmental niches of Karachi, Pakistan. Obtained results revealed the highest prevalence of E. faecium and E. faecalis in all environments. Bacterial antagonism and enterocin genes were observed significantly high in poultry environments. The inter-environmental correlation indicated a strong positive correlation of freshwater with sewage and soil environments. Similarly, the fsr regulatory system was mostly identified in poultry-related environments, and a significant correlation between QS system and biofilm formation was established. In conclusion, this study confirmed the high prevalence of E. faecium in all tested sources, high enterocin production in non-clinical environments, and more fsr regulatory genes in poultry-related environments.

Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration, and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the central composite design. The strain produced 20 IU mL-1 of pectinase after 24 h under optimized conditions. The enzyme production kinetics data also confirmed that 24 h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of >65 kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations.

Enzyme Profiling and Identification of Endophytic and Rhizospheric Bacteria Isolated from Arthrocnemum macrostachyum.

Endophytic and rhizospheric bacteria isolated from halophytic plants support their host to survive in hyper-saline soil. These bacteria are also known to produce various enzymes with potential industrial applications. In this study, the endophytic and rhizospheric bacteria were isolated from Arthrocnemum macrostachyum collected from Karachi, Pakistan, and their ability to produce various extracellular enzymes was assessed using commercial and natural substrates. In total, 11 bacterial strains were isolated (four endophytic; seven rhizospheric). Bacillus was found to be the most abundant genus (73%), followed by Glutamicibacter (27%). The isolates including Glutamicibacter endophyticus and Bacillus licheniformis are reported for the first time from A. macrostachyum. All of the isolates were capable of producing at least two of the five industrially important hydrolytic enzymes tested, i.e., xylanase, cellulase, amylase, pectinase, and lipase. Lipase production was found to be highest among the isolates, i.e., up to 18 IU mL-1. Although most of the isolates could grow at a wide range of temperatures (4-55 °C), pH (1-11), and salt concentrations (2-12%), under extreme conditions, very little growth was observed and the optimal growth was recorded between 2% and 6% NaCl, 25 and 45 °C, and 7 and 9 pH. Our results suggest that these isolates could be potential producers of enzymes with several biotechnological applications.

Evaluation of heavy metal pollutants in salt and seawater under the influence of the Lyari River and potential health risk assessment.

Heavy metals intoxication through edible salt poses serious health hazards. The conducted research assessed the levels of heavy metals, health risks of salt samples, and the pollution index of seawater obtained from saltpans located at Sandpit, Karachi, Pakistan, which receive untreated effluents through the Lyari River. Seawater (n = 27) and salt samples (n = 27) were prepared for the detection of Al, Cd, Pb, Cr, Fe, Cu, Hg, Ni, As and Zn through atomic absorption spectroscopy, mean concentrations ± S.D. (mg/mL) were compared with the National Environmental Quality Standards, Pakistan. The levels of Cr (40.06 ± 0.21) were the highest followed by Fe (39.77 ± 0.08) > As (25.12 ± 0.21) > Ni > Pb > Al > Hg > Zn > Cd > Cu. In sea salt (Mean ± S·D mg/Kg), the Cr (47.79 ± 0.19), Fe (47.5 ± 0.15), As (30.62 ± 0.22), and Ni were redundant elements followed by Al > Pb > Hg > Zn > Cu > Cd. The water comprehensive pollution index was 1000 times greater than the critical values. The single factor pollution index was highest for Hg (1321), followed by Cr (40), Ni (36), Pb (32), Al (31.4), Cd (31.3), and As (25). Health risk assessment indices (from salt samples), including target hazard quotient (THQ) for As, Pb, Al, Hg, and Cr were two to six times higher than the noncarcinogenic THQ reference range. Similarly, the hazardous index indicated that salt was 20 times hazardous (HI = 20.29), and the carcinogenic rate index for the heavy metals i.e., Cd, As, Cr, and Ni was above the reference CR value i.e., 1 × 10-4. In conclusion, the experimental and theoretical approaches recommend that the use of contaminated salt may impart health hazards.

Ionic strength effects on the photodegradation reactions of riboflavin in aqueous solution.

A study of the effect of ionic strength on the photodegradation reactions (photoreduction and photoaddition) of riboflavin (RF) in phosphate buffer (pH7.0) has been carried out using a specific multicomponent spectrometric method. It has been found that the rates of photodegradation reactions of RF are dependent upon the ionic strength of the solutions at different buffer concentrations. The apparent first-order rate constants (kobs) for the photodegradation of riboflavin at ionic strengths of 0.1-0.5 (0.5M phosphate) lie in the range of 7.35-30.32 × 10(-3) min(-1). Under these conditions, the rate constants for the formation of the major products, lumichrome (LC) by photoreduction pathway, and cyclodehydroriboflavin (CDRF) by photoaddition pathway, are in the range of 3.80-16.03 and 1.70-6.07 × 10(-3) min(-1), respectively. A linear relationship has been observed between log kobs and √µ/1+√µ. A similar plot of log k/ko against √µ yields a straight line with a value of ~+1 for ZAZB showing the involvement of a charged species in the rate determining step. NaCl appears to promote the photodegradation reactions of RF probably by an excited state interaction. The implications of ionic strength on RF photodegradation by different pathways and flavin-protein interactions have been discussed.