Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Production and structural characterization of eco-friendly bioemulsifier SC04 from Saccharomyces cerevisiae strain MYN04 with potential applications.

BACKGROUND: Bioemulsifiers are natural or microbial-based products with the ability to emulsify hydrophobic compounds in water. These compounds are biodegradable, eco-friendly, and find applications in various industries. RESULTS: Thirteen yeasts were isolated from different sources in Alexandria, Egypt, and evaluated for their potential to produce intracellular bioemulsifiers. One yeast, isolated from a local market in Egypt, showed the highest emulsification index (EI24) value. Through 26S rRNA sequencing, this yeast was identified as Saccharomyces cerevisiae strain MYN04. The growth kinetics of the isolate were studied, and after 36 h of incubation, the highest yield of cell dry weight (CDW) was obtained at 3.17 g/L, with an EI24 of 55.6%. Experimental designs were used to investigate the effects of culture parameters on maximizing bioemulsifier SC04 production and CDW. The study achieved a maximum EI24 of 79.0 ± 2.0%. Furthermore, the crude bioemulsifier was precipitated with 50% ethanol and purified using Sephadex G-75 gel filtration chromatography. Bioemulsifier SC04 was found to consist of 27.1% carbohydrates and 72.9% proteins. Structural determination of purified bioemulsifier SC04 was carried out using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance spectroscopy (NMR). FTIR spectroscopy revealed characteristic bands associated with carboxyl and hydroxyl groups of carbohydrates, as well as amine groups of proteins. HPLC analysis of monosaccharide composition detected the presence of mannose, galactose, and glucose. Physicochemical characterization of the fraction after gel filtration indicated that bioemulsifier SC04 is a high molecular weight protein-oligosaccharide complex. This bioemulsifier demonstrated stability at different pH values, temperatures, and salinities. At a concentration of 0.5 mg/mL, it exhibited 51.8% scavenging of DPPH radicals. Furthermore, in vitro cytotoxicity evaluation using the MTT assay revealed a noncytotoxic effect of SC04 against normal epithelial kidney cell lines. CONCLUSIONS: This study presents a new eco-friendly bioemulsifier, named SC04, which exhibits significant emulsifying ability, antioxidant and anticancer properties, and stabilizing properties. These findings suggest that SC04 is a promising candidate for applications in the food, pharmaceutical, and industrial sectors.

Bioremediation of kerosene I: A case study in liquid media.

The ability of different local isolates in addition to some isolates from Germany to degrade kerosene in liquid medium was studied. The results showed that the percent of kerosene degradation varied among the different organisms and that 59-94% of kerosene was degraded after 21d. Two local isolates (Pseudomonas sp. AP and Pseudomonas sp. CK) and one German isolate (Gordonia sp. DM) were selected for this study. The addition of wheat bran, as co-substrate, stimulated the kerosene degradation by the two local strains, while glucose inhibited the degradation rate using the three organisms with different rates. Ammonium nitrate and urea was the best nitrogen sources. The use of superphosphate (as phosphorus source) in the presence of urea stimulates the degradation rate. It was also observed that the addition of 1% surfactants, like Triton X-100, Igepal, Tergitol, or Tween 20 and 80 enhanced the kerosene degradation. The degradation percent lied between 94% and 98%. The ability of the tested organisms to degrade kerosene concentration from 2% to 8% was evaluated. It was found that the three organisms degraded about 65-85% from 8% kerosene after 21d. The use of rice straw-immobilized cells reduced the time of degradation and enhanced the degradation ability of the organisms. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the presence of a common protein band when the tested organisms were grown on kerosene.

Optimization and purification of alkaline proteases produced by marine Bacillus sp. MIG newly isolated from eastern harbour of Alexandria.

A marine Bacillus strain was isolated from the eastern harbour of Alexandria and identified as Bacillus sp. MIG. Maximum activity of studied proteases was obtained when the bacterium was grown in medium with 1% wheat bran and 0.5% yeast extract in addition to the mineral salts and incubated for 48 h at 30 degrees C and 120 rpm. Two alkaline proteases (Pro 1 and Pro 2) were purified to homogeneity using cation exchange chromatography on CM-Sepharose CL-6B followed by Sephadex G-75 superfine. The optimum activities were at pH 11 or 12, and temperatures of 50 and 55 degrees C for Pro 1 and Pro 2 respectively. These two enzymes were relatively stable over pH range from 7.0- 11. Pro 2 was found to be more stable at 50 degrees C in absence of Ca2+ and retained about 47% of its activity after 3 h at this temperature, while Pro 1 lost its activity completely at the same conditions. The two enzymes were active against haemoglobin and casein; in addition, Pro 2 exhibited moderate activity against keratin. Both enzymes were partially inhibited by Ag+ and Hg2+. PMSF completely inhibited the enzymes, while dithiothreitol and 2-mercaptoethanol stimulated their activities, suggesting to be thiol-dependent serine proteases. The enzymes were stable in the presence of the surfactants and bleaching agent (H2O2) and relatively stable in presence of some commercial detergents.

Catalytic properties of the immobilized Aspergillus tamarii xylanase.

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher Km (Michaelis constant), lower Vmax (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60 degrees C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60 degrees C for the immobilized enzyme is about 6.0 x 10(-3), which is lower than that of the free enzyme (7.77 x 10(-3) min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.

Production of a polyester degrading extracellular hydrolase from Thermomonospora fusca.

The production of a polyester-degrading hydrolase from the thermophilic actinomycete Thermomonospora fusca was investigated with regard to its potential technical application. Only in the presence of a polyester (random aliphatic-aromatic copolyester from 1,4-butanediol, terephthalic acid, and adipic acid with around 40-50 mol % terephthalic acid in the acid component), the excretion of the extracellular enzyme could be achieved with an optimized synthetic medium using pectin and NH(4)Cl as nitrogen source. Compared to complex media, a significantly higher specific activity at comparable volumetric yields could be obtained, thus reducing the expenditure for purification. The activity profile in the medium is controlled by a complex process involving (1) induction of enzyme excretion, (2) enzyme adsorption on the hydrophobic polyester surface, (3) inhibition of enzyme generation by monomers produced by polyester cleavage, and (4) enzyme denaturation. Diafiltration with cellulose acetate membranes as the sole downstream processing step led to a product of high purity and with sufficient yield (60% of total activity). Scaling-up from shaking flasks to a fermentor scale of 100 L revealed no specific problems. However, the excretion of the hydrolase by the actinomycete turned out to be inhibited by the degradation products (monomers) of the aliphatic-aromatic copolyester used as inductor for the enzyme production. The crude enzyme exhibited generally similar properties (temperature and pH optimum) as the highly purified hydrolase described previously; however, the storage capability and thermal stability is improved when the crude enzyme solution is diafiltrated.