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Biochimie Feb 2016Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this...
Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this enzymatic drug is responsible for several life threatening side effects. This study describes the purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis. The purified enzyme exhibited molecular mass of approximately 205±3 kDa on native-PAGE and ̴34±1 kDa on SDS-PAGE, revealing that the enzyme is homohexamer. The isoelectric point of enzyme was 5.5, calculated by 2D-PAGE. Optimum activity of asparaginase was achieved at 40 °C and pH 7.5, which is close to the internal environment of the human body. Monovalent cations (Na(+) and K(+)) and reducing agents (2-mercaptoethanol and glutathione) has enhanced asparaginase activity. Whereas, divalent (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)), trivalent (Fe(3+)) cations and thiol group blocking agent (iodoacetamide) inhibited the enzyme activity significantly. In vitro serum and trypsin half life of asparaginase is almost 2 and 1.5 fold respectively, which is higher than commercial asparaginase. MTT assay results showed that the anticancer activity of purified asparaginase was comparable or higher than commercial E. coli asparaginase. Microscopic studies and cell cycle analysis suggested that purified enzyme induced apoptotic cell death in dose-dependent manner. Loss of mitochondrial membrane potential suggests that enzyme induces cell death via activation of intrinsic apoptotic pathway. Purified asparaginase was found to be nontoxic for human noncancerous FR-2 cells and human blood lymphocytes, which is a remarkable therapeutic feature.
Topics: Apoptosis; Asparaginase; Cell Cycle; Electrophoresis, Polyacrylamide Gel; Glutaminase; Humans; Pseudomonas
PubMed: 26597158
DOI: 10.1016/j.biochi.2015.11.012 -
Microbiology Resource Announcements Mar 2024Here, we report the draft genome sequences of strains zfem001-005, five isolates from the intestinal microbiota of healthy larval zebrafish at a developmental age of 7...
Here, we report the draft genome sequences of strains zfem001-005, five isolates from the intestinal microbiota of healthy larval zebrafish at a developmental age of 7 days post fertilization. The isolates have been identified as , , , , and , respectively.
PubMed: 38376340
DOI: 10.1128/mra.00934-23 -
The Journal of Antimicrobial... Aug 2022There is no comprehensive study on PAM-like MBLs.
BACKGROUND
There is no comprehensive study on PAM-like MBLs.
OBJECTIVES
Our aim was to characterize novel B3 MBL variants, PAM-2 and PAM-3, from Pseudomonas tohonis clinical isolates.
METHODS
We evaluated the antimicrobial susceptibility and the MBL gene composition of three novel P. tohonis clinical isolates identified at a Japanese hospital, using the broth microdilution method and WGS, respectively. We characterized the PAM-2 and PAM-3 proteins using recombinant protein expression and biochemical evaluations.
RESULTS
Low carbapenem MICs (meropenem MIC = 0.125-1 mg/L) were observed for all three P. tohonis isolates; however, the isolates produced MBLs. We identified blaPAM-2 and blaPAM-3 as potential genes, belonging to a novel subclass of B3 MBLs. Their genomic sequence was similar to that of blaPAM-1 from Pseudomonas alcaligenes. PAM-2 and PAM-3 comprised 287 amino acids and exhibited 90% amino acid identity with PAM-1, 73% identity with POM-1 from Pseudomonas otitidis and 61% identity with L1 from Stenotrophomonas maltophilia. Biochemical evaluations of recombinant PAM-2 and PAM-3 revealed similar kcat/Km ratios and demonstrated catalytic activity against all the tested β-lactams, except for aztreonam. In addition, the kcat/Km ratio for imipenem was 40-fold lower than that for meropenem.
CONCLUSIONS
P. tohonis harbours a species-specific PAM-family MBL gene. This enzyme has higher hydrolytic activity against meropenem compared with that against imipenem.
Topics: Anti-Bacterial Agents; Humans; Imipenem; Meropenem; Microbial Sensitivity Tests; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; beta-Lactamases
PubMed: 35786775
DOI: 10.1093/jac/dkac210 -
World Journal of Microbiology &... Aug 2020The study aimed to investigate the efficiency of exogenous bacterial consortium (Enterobacter cloacae and Pseudomonas otitidis) decorated (immobilized) with FeO...
The study aimed to investigate the efficiency of exogenous bacterial consortium (Enterobacter cloacae and Pseudomonas otitidis) decorated (immobilized) with FeO Nanoparticles for the treatment of petroleum hydrocarbon-contaminated wastewater. Glycine coated magnetite Nanoparticles (FeO NPs) were prepared using reverse co-precipitation method and were characterized using X-ray diffraction, transmission and scanning electron microscopy and vibrating sample magnetometer. They were used to decorate exogenous bacterial consortium (Enterobacter cloacae and Pseudomonas otitidis) at 3 different FeO/bacteria ratios (1:1, 1:3 and 3:1 w/w). Bioremediation of oil contaminated wastewater collected from one of the petroleum distribution companies, Alexandria was conducted for 168 h using FeO/bacterial association at the best ratio (3:1) and compared with non-decorated consortium and the indigenous bacteria in the control. Analysis indicated crystalline structure of FeO NPs with spherical particles (size: 15-20 nm) and superparamagnetic properties. Glycine modified-FeO exhibited high ability to immobilize bacteria which acquired its magnetic properties. The highest coating efficiency (92%) was achieved at 3:1 FeO/bacteria ratio after 1 h. This ratio positively affected bacterial growth reaching the highest growth rate (5.07 fold higher than the control) after 4 h. The highest removal efficiencies of the total suspended solids (TSS), chemical oxygen demands (COD), oil and grease (O&G) and total petroleum hydrocarbons (TPH) recording 96, 65.4, 83.9 and 85% reaching residual concentrations of 9.5, 598, 99 and 60 mg/l respectively were achieved after 4 h by the FeO-bacteria assembly. Compared with the maximum permissible limits of the tested parameters, TSS residue was highly compiled with its limit (50 mg/l), while COD, O&G and TPH were 7.5, 9.9, and 120-folds higher than their limits (100, 15 and 0.5 mg/l respectively). To the best of our knowledge it is first time to use integrated Enterobacter cloacae and Pseudomonas otitidis consortium decorated with FeO NPs for the treatment of petroleum hydrocarbon-contaminated wastewater. The proposed system proved to be a very efficient, economical and applicable for the removal of the included contaminants in very short running time which increases its biotechnological added value.
Topics: Bacteria; Biodegradation, Environmental; Biological Oxygen Demand Analysis; Ferrosoferric Oxide; Hydrocarbons; Immobilization; Magnetite Nanoparticles; Microbial Consortia; Nanoparticles; Petroleum; Pseudomonas; Wastewater; Water Purification; X-Ray Diffraction
PubMed: 32813039
DOI: 10.1007/s11274-020-02915-1 -
Water Environment Research : a Research... Dec 2019More than 2.1 billion people worldwide are deprived of safe drinking water at homes. The situation is strikingly worse in a developing country like Pakistan where over...
More than 2.1 billion people worldwide are deprived of safe drinking water at homes. The situation is strikingly worse in a developing country like Pakistan where over 69% of the population does not have access to safe drinking water. The present study evaluated a perenial herb, Typha angustata (TA), to purify the spring water. For this purpose, 25 water samples were collected. Majority of samples (20/25) were highly contaminated with microbes ranging colony forming units (CFU) per millileter per Petri dish ranged from 85 to 279 with an average of 136.4. Microbial inhibition of water samples treated with the nonmodified plant extract was observed to be better with the average of 55.5% compared to the treatment with NaOH chemically modified plant where average 46.4% inhibition of microbial load was observed. Four species of microbes such as Bacillus cereus, Pseudomonas aeruginosa, Pseudomonas otitidis, and Streptococcus agalactiae were identified after sequencing. We concluded that T. angustata extract may be used as an antibacterial agent/biosorbent for the purification of drinking water to provide safe drinking water to billions of humans. PRACTITIONER POINTS: Spring water samples were collected from 25 different springs. Spring water samples were analyzed for physiochemical parameters. Spring water samples were found to be contaminated with pathogenic bacteria, that is, Bacillus cereus, Pseudomonas aeruginosa, Pseudomonas otitidis, and Streptococcus agalactiae. Pathogenic bacteria in spring water samples were treated with extract of Typha angustata. Extract of Typha angustata was found as a potential antibacterial agent against pathogenic bacteria.
Topics: Bacteria; Humans; Pakistan; Typhaceae; Water Microbiology; Water Supply
PubMed: 31306534
DOI: 10.1002/wer.1182 -
Journal of Global Antimicrobial... Jun 2020The presence of carbapenemase-producing bacterial isolates is found not only in hospital and community settings but also in the environment. Carbapenemase production may...
OBJECTIVES
The presence of carbapenemase-producing bacterial isolates is found not only in hospital and community settings but also in the environment. Carbapenemase production may be related to acquired, usually plasmid-borne, β-lactamase genes or to chromosomal genes intrinsic to various species. The aim of this study was to evaluate the occurrence of such carbapenemase-producing bacterial isolates among environmental samples from Nigeria.
METHODS
A total of 122 environmental samples were plated on carbapenem-containing media. A total of 259 isolates were recovered, among which 124 were carbapenemase-producers according to the results of the Rapidec® Carba NP test.
RESULTS
The majority of isolates (n=112) recovered corresponded to natural producers of carbapenemases, i.e. Stenotrophomonas maltophilia (n=108), Burkholderia cepacia (n=1), Shewanella sp. (n=1), Sphingobacterium sp. (n=1) and Chryseobacterium gleum (n=1). Ten isolates (mainly Enterobacteriaceae and Acinetobacter baumannii) produced an acquired carbapenemase, most commonly of the NDM type. In addition, two Pseudomonas otitidis isolates were identified as producing the Ambler class B carbapenemase POM-1, further confirming that this carbapenemase is naturally produced in this environmental species. Finally, several isolates co-producing 16S rRNA methylases (ArmA, RmtC) and/or extended-spectrum β-lactamases (CTX-M-9, CTX-M-15) were also identified.
CONCLUSION
This study revealed the presence and diversity of clinically-relevant antimicrobial-resistant bacteria in the environment in Nigeria.
Topics: Bacterial Proteins; Chryseobacterium; Nigeria; Pseudomonas; RNA, Ribosomal, 16S; beta-Lactamases
PubMed: 31639547
DOI: 10.1016/j.jgar.2019.10.014 -
Ecotoxicology and Environmental Safety Jan 2023Nafion by-product 2 (Nafion BP2), an emerging fluorinated sulfonic acid commonly used in polymer electrolyte membrane technologies, has been detected in various...
Nafion by-product 2 (Nafion BP2), an emerging fluorinated sulfonic acid commonly used in polymer electrolyte membrane technologies, has been detected in various environmental and human matrices. To date, however, few studies have explored its toxicity. In this study, zebrafish embryos were exposed to Nafion BP2 at concentrations of 20, 40, 60, 80, 100, 120, 140, and 160 mg/L from fertilization to 120 post-fertilization (hpf), and multiple developmental parameters (survival rate, hatching rate, and malformation rate) were then determined. Results showed that Nafion BP2 exposure led to a significant decrease in survival and hatching rates and an increase in malformations. The half maximal effective concentration (EC) of Nafion BP2 for malformation at 120 hpf was 55 mg/L, which is higher than the globally important contaminant perfluorooctane sulfonate (PFOS, 6 mg/L). Furthermore, exposure to Nafion BP2 resulted in additional types of malformations compared to PFOS exposure. Pathologically, Nafion BP2 caused abnormal early foregut development, with exfoliation of intestinal mucosa, damage to lamina propria, and aberrant proliferation of lamina propria cells. Nitric oxide content also decreased markedly. In addition, embryos showed an inflammatory response following Nafion BP2 exposure, with significantly increased levels of pro-inflammatory factors C4 and IL-6. Acidic mucin in the hindgut increased more than two-fold. 16 S rRNA sequencing revealed a marked increase in the pathogen Pseudomonas otitidis. Furthermore, pathways involved in intestinal protein digestion and absorption, inflammatory response, and immune response were significantly altered. Our findings suggest that the intestine is a crucial toxicity target of Nafion BP2 in zebrafish, thus highlighting the need to evaluate its health risks.
Topics: Animals; Humans; Embryo, Nonmammalian; Fluorocarbon Polymers; Homeostasis; Intestines; Water Pollutants, Chemical; Zebrafish
PubMed: 36508837
DOI: 10.1016/j.ecoenv.2022.114368 -
Diagnostic Microbiology and Infectious... May 2007Laboratory detection of Pseudomonas spp., particularly Pseudomonas aeruginosa, is an important assay in the nosocomial control. The study was designed firstly to...
Laboratory detection of Pseudomonas spp., particularly Pseudomonas aeruginosa, is an important assay in the nosocomial control. The study was designed firstly to establish a new assay-applied LightCycler polymerase chain reaction (PCR) technology with melting curve analysis (MCA). A total of 224 Gram-negative isolates were used to verify the assay system. The PCR with MCA method using the P. aeruginosa-specific gyrase B gene primers was rapid and accurate; the total run is approximately 3 h, and the sensitivity and specificity relative to the Vitek (bioMerieux, Hazelwood, MO) results were 98.1% and 100%, respectively. Vitek identification system was not able to identify the isolates from the new Pseudomonas otitidis spp. opposite to the real-time PCR. This assay was validated to be accurate with an overall sensitivity and specificity of 98.7% and 98.9%, respectively. Conclusively, this rapid and accurate PCR assay with MCA will help to manage and control infections with P. aeruginosa.
Topics: Bacterial Typing Techniques; Base Sequence; DNA Gyrase; DNA Primers; DNA, Bacterial; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Pseudomonas Infections; Pseudomonas aeruginosa; Sensitivity and Specificity; Time Factors; Transition Temperature
PubMed: 17368797
DOI: 10.1016/j.diagmicrobio.2006.11.007 -
Protoplasma Jul 2016Thermoplastic-based materials are recalcitrant in nature, which extensive use affect environmental health. Here, we attempt to compare the response of indigenously...
Thermoplastic-based materials are recalcitrant in nature, which extensive use affect environmental health. Here, we attempt to compare the response of indigenously produced bacterial consortium-I and consortium-II in degrading polyvinyl chloride (PVC). These consortia were developed by using different combination of bacterial strains of Pseudomonas otitidis, Bacillus cereus, and Acanthopleurobacter pedis from waste disposal sites of Northern India after their identification via 16S rDNA sequencing. The progressive degradation of PVC by consortia was examined via scanning electron microscopy, atomic force microscopy, UV-vis, FT-IR spectra, gel permeation chromatography, and differential scanning calorimetry analysis at different incubations and time intervals. The consortium-II was superior over consortium-I in degrading the PVC. Further, the carbon source utilization analysis revealed that the extensive use of consortia has not any effect on functional diversity of native soil microbes.
Topics: Biodegradation, Environmental; Microbial Consortia; Molecular Typing; Polyvinyl Chloride; RNA, Ribosomal, 16S; Soil Microbiology; Soil Pollutants
PubMed: 26231814
DOI: 10.1007/s00709-015-0855-9 -
International Health Sep 2020The present study was carried out to investigate the tap water quality of public toilets in Amritsar, Punjab, India.
BACKGROUND
The present study was carried out to investigate the tap water quality of public toilets in Amritsar, Punjab, India.
METHODS
Water samples from the taps of the public toilets were collected in sterile containers and physicochemical and bacteriological analysis was performed using standard methods. Also, genotypic and phenotypic characterization of the bacterial isolates was performed using different biochemical tests and 16S ribosomal RNA analysis. An antibiotic susceptibility test was performed using antibiotics based on their mode of action. A biofilm assay was performed to assess the adhesion potential of the isolates.
RESULTS
A total of 25 bacterial isolates were identified from the water samples, including Acinetobacter junii, Acinetobacter pittii, Acinetobacter haemolyticus, Bacillus pumilus, Bacillus megaterium, Bacillus marisflavi, Bacillus flexus, Bacillus oceanisediminis, Pseudomonas otitidis, Pseudomonas sp. RR013, Pseudomonas sp. RR021, Pseudomonas sp. RR022, Escherichia coli and Enterobacter cloacae. The results of the antimicrobial susceptibility test revealed that the antibiotics cefodroxil, aztreonam, nitrofurantoin, cefepime, ceftazidime and amoxyclav were found to be mostly ineffective against various isolates. The biofilm assay revealed the weak, moderate and strong biofilm producers among them.
CONCLUSIONS
The tap water in the public toilets was microbially contaminated and needs to be monitored carefully. The antibiotic susceptibility profile showed that of 25 bacterial isolates, 5 were multidrug resistant. Bacterial isolates exhibited strong to weak adhesion potential in the biofilm assay.
Topics: Acinetobacter; Anti-Bacterial Agents; Bacillus; Bacterial Infections; Bathroom Equipment; Biofilms; Genotype; Humans; India; Microbial Sensitivity Tests; Phenotype; Pseudomonas; Water; Water Microbiology
PubMed: 31693132
DOI: 10.1093/inthealth/ihz074