scholarly journals Detection of Polymorphism in the Gene blaKPC2 of Local Klebsiella pneumoniae Isolated from Iraqi Patients

2017 ◽  
Vol 11 (1) ◽  
pp. 26-33
Author(s):  
Saadi Abd-Alkareem Jasim ◽  
Mohammed Maaroof ◽  
Najwa Ahmed
Keyword(s):  
Environmental Management ◽  
Klebsiella Pneumoniae ◽  
Dna Sequences ◽  
Microscopic Examination ◽  
Gene Bank ◽  
Bacterial Isolates ◽  
Wild Type ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Severe Infections

Carbapenemases are clinically important because they destroy and may confer a resistance to carbapenems, severe infections caused by carbapenemase producers is associated with increased mortality. To achieve this goal, 180 samples were collected from different clinical sources included 92 urine, 33 smears of wounds, 13 smears of burns and 42 sputum. The samples were taken from patients attended Al-Yarmouk Teaching Hospital and Ibin Baladi Hospital in Baghdad Governorate. Diagnosis of bacterial isolates was done depending upon the microscopic examination, the cultured characteristics and biochemical tests. DNA extracted from 84 samples. Accordingly, detection of blaKPC2 gene was conducted by using specific primers for amplification of blaKPC2 gene. Moreover, the sequencing of 910 bp for blaKPC2 gene was performed by the biotechnology lab. at the National Instrumentation Center for Environmental Management (NICEM). Such test has been implanted by using 3730XL as a DNA sequences. The obtained results were analyzed by blast at the National Center Biotechnology Information (NCBI) and detect polymorphism in blaKPC2 based on the Bio Edit. Consequently, 94 variations between 47 transversions, 43 transitions and 4 deletions nucleotide were noticed. In a sense the test showed 79% under sequence ID gb|CP009872.1| from 3037673 -3038166 number of nucleotide from K. pneumoniae subsp. pneumoniae strain KPNIH30 of Gene Bank, score (329) and expect 5e-86 with the wild type of blaKPC2 gene from Gene Bank. Finally, the results illustrated polymorphism between local strains of

scholarly journals The Discovery of Vibrio harveyi on Litopenaeus vannamei Infected White Feces Disease in Situbondo, East Java

2020 ◽  
Vol 22 (1) ◽  
pp. 9
Author(s):  
Sumini Sumini ◽  
Rahayu Kusdarwati
Keyword(s):  
Water Quality ◽  
Vibrio Harveyi ◽  
Total Organic Matter ◽  
Bacterial Isolates ◽  
Abnormal Result ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Poor Water Quality ◽  
Brackishwater Pond ◽  
Test Result

This research was conducted to discover Vibrio harveyi infected vannamei shrimp with White Feces Disease (WFD) in Situbondo, East Java Province. This research was conducted in November 2017 until September  2018. All Vibrio isolates from shrimp infected WFD were identified with biochemical tests, Analytical Profile Index/ API 20NE (BioMeriuex), and PCR with specific primers for V. harveyi. Additional parameters were the water quality, plankton brackishwater pond abundance, and antibiotic resistance test. Result showed that from 17 bacterial isolates identified, 10 isolates were V. harveyi (58.82%), three isolates were V. alginolyticus (17.65%), one V. fluvialis isolate (5.88%), one V. parahaemolyticus isolate (5.88%), and two non Vibrio isolates which were identified as Shewanella putrefaciens (11.76%). All isolates of V. harveyi also showed resistance activity on more than one antibiotic. Poor water quality had been identified as abnormal result of pH, alkalinity, salinity, ammonia levels and total organic matter level. Plankton abundance observation showed that Chloropyceae, Diatom, and Dinoflagellata dominated all sampled brackishwater ponds. This research concluded that V. harveyi were discovered on vannamei shrimp with poor water quality and plankton abundance in the pond samples.

scholarly journals Susceptibility of Klebsiella Pneumoniae Isolated from Pus Specimens of Post-Surgery Patients in Medan, Indonesia to Selected Antibiotics

2019 ◽  
Vol 7 (22) ◽  
pp. 3861-3864 ◽  
Author(s):  
Popi Patilaya ◽  
Dadang Irfan Husori ◽  
Lany Marhafanny
Keyword(s):  
Klebsiella Pneumoniae ◽  
Klebsiella Pneumonia ◽  
Good Sensitivity ◽  
Bacterial Isolates ◽  
Inhibitory Zone ◽  
Biochemical Tests ◽  
Post Surgery ◽  
Low Sensitivity

AIM: This study was to determine the sensitivity of Klebsiella pneumonia isolated from pus specimens of post-surgery patients in Medan, Indonesia to selected antibiotics. METHODS: Samples were collected at the Laboratory of Microbiology, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia. The isolated bacteria were identified by Gram’s stain, colony characteristics, and biochemical tests. Susceptibility of K. pneumoniae isolates were tested to selected antibiotics including amikacin, meropenem, levofloxacin, ciprofloxacin, co-trimoxazole, ceftazidime, cefoperazone, cefuroxime, cefepime, cefotaxime, tetracycline, chloramphenicol, amoxicillin and ampicillin with Kirby Bauer method by measuring the inhibitory zone. RESULTS: A total of 20 K. pneumoniae isolates were obtained in this study. The results showed that K. Pneumonia isolates exhibited good sensitivity to amikacin (100%) and meropenem (80%). Sensitivity of levofloxacin (60%), ceftazidime (55%), ciprofloxacin (55%), cefoperazone (50%), and co-trimoxazole (50%) were moderate for the bacterial isolates. K. Pneumoniae isolates indicated low sensitivity to cefuroxime (45%), chloramphenicol (35%), cefepime (30%), cefotaxime (30%), tetracycline (30%), amoxicillin (5%), and ampicillin (5%). CONCLUSION: This study concludes that K. pneumoniae isolates are most sensitive to amikacin and less sensitive to ampicillin and amoxicillin.

Identification Pseudomonas aeruginosa by OprD Gene for Differentiation from Other Pseudomonas Species that Isolated from Clinical Samples

2019 ◽  
Vol 9 (01) ◽  
pp. 46-50
Author(s):  
Ashwak B Al-Hashimy ◽  
Huda S Alagely ◽  
Akeel K Albuaji ◽  
Khalid R Majeed
Keyword(s):  
Pseudomonas Aeruginosa ◽  
General Hospital ◽  
Culture Media ◽  
Final Diagnosis ◽  
Clinical Samples ◽  
Bacterial Isolates ◽  
Molecular Method ◽  
Biochemical Tests ◽  
Pseudomonas Species ◽  
Specific Sequences

The present study included the collection of 100 samples from various clinical sources for investigating the presence of P. aeruginosa in those sources, the samples have been collected from some hospitals in Baghdad and Hillah city (Al-qassim General Hospital, ,Al-hillah teaching hospital,and Al-hashimya General hospital ) which included wounds, burns, ear and sputum infections. The study was carried out through October 2017 till the end of March 2018. The samples were identified based on the morphological and microscopically characteristics of the colonies when they were culturing or number of culture media as well as biochemical tests, molecular identification were also used as a final diagnostic test for isolates that were positive as they belong to P.aeruginosa bacteria during previous tests based on the OprD gene which has specific sequences for P.aeruginosa bacteria as a detection gene and also consider as virulence factor so it have a synonyms mechanism to antibiotic resistance . The results of the final diagnosis showed that 38 isolates belong to target bacteria were distributed as 18 of burns, 11 isolates of wounds, 6 isolates of ear infection and 3 isolates of sputum, The examination of the sensitivity of all bacterial isolates was done for elected 38 isolation towards the 9 antibiotic by a Bauer - Kirby and the isolates were resistant for a number of antibiotics used such as Ciprofloxacin 65.7%, Norflaxacin 71%, Imipenem 63.1% Meropenem 68.4%, Gentamicin 65.7%, Amikacin 26.3%, Cefepime 68.4%, Ceftazidime 65.7% and Piperacillin 57.8%.Molecular method , All isolates (38) of P. aeruginosa positive for the diagnostic special gene (OprD) genes (100%).

Phylogeny Characterization of Seb Gene Encoding Enterotoxins in Staphylococcus aureus Isolated from Raw Milk and Cheese

2019 ◽  
Vol 9 (o3) ◽  
Author(s):  
Fatima N. Aziz ◽  
Laith Abdul Hassan Mohammed-Jawad
Keyword(s):  
Staphylococcus Aureus ◽  
Food Poisoning ◽  
Raw Milk ◽  
Staphylococcal Enterotoxin B ◽  
Human Pathogen ◽  
Conventional Pcr ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Gene Encoding

Food poisoning due to the bacteria is a big global problem in economically and human's health. This problem refers to an illness which is due to infection or the toxin exists in nature and the food that use. Milk is considered a nutritious food because it contains proteins and vitamins. The aim of this study is to detect and phylogeny characterization of staphylococcal enterotoxin B gene (Seb). A total of 200 milk and cheese samples were screened. One hundred ten isolates of Staphylococcus aureus pre-confirmed using selective and differential media with biochemical tests. Genomic DNA was extracted from the isolates and the SEB gene detects using conventional PCR with specific primers. Three staphylococcus aureus isolates were found to be positive for Seb gene using PCR and confirmed by sequencing. Sequence homology showed variety range of identity starting from (100% to 38%). Phylogenetic tree analyses show that samples (6 and 5) are correlated with S. epidermidis. This study discovered that isolates (A6-RLQ and A5-RLQ) are significantly clustered in a group with non- human pathogen Staphylococcus agnetis.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

2021 ◽  
Vol 9 (2) ◽  
pp. 335
Author(s):  
Novaria Sari Dewi Panjaitan ◽  
Yu-Tze Horng ◽  
Chih-Ching Chien ◽  
Hung-Chi Yang ◽  
Ren-In You ◽  
...  
Keyword(s):  
Survival Rate ◽  
Klebsiella Pneumoniae ◽  
Laser Scanning ◽  
Bacterial Resistance ◽  
Capsular Polysaccharide ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Bacteria ◽  
Wild Type ◽  
Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

scholarly journals Uncommon Non-Candida Yeasts in Healthy Turkeys—Antimicrobial Susceptibility and Biochemical Characteristic of Trichosporon Isolates

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 538
Author(s):  
Kamila Bobrek ◽  
Ireneusz Sokół ◽  
Andrzej Gaweł
Keyword(s):  
Gastrointestinal Tract ◽  
Amphotericin B ◽  
Antimicrobial Susceptibility ◽  
Antifungal Susceptibility ◽  
Range Limit ◽  
Biochemical Tests ◽  
Lower Range ◽  
Normal Microflora ◽  
Severe Infections ◽  
Diverse Community

The microbiota of the gastrointestinal tract of humans and animals is inhabited by a diverse community of bacteria, fungi, protozoa, and viruses. In cases where there is an imbalance in the normal microflora or an immunosuppression on the part of the host, these opportunistic microorganisms can cause severe infections. The study presented here evaluates the biochemical and antifungal susceptibility features of Trichosporon spp., uncommon non-Candida strains isolated from the gastrointestinal tract of healthy turkeys. The Trichosporon coremiiforme and Trichosporon (Apiotrichum) montevideense accounted for 7.7% of all fungi isolates. The biochemical tests showed that Trichosporon coremiiforme had active esterase (C4), esterase-lipase (C8) valine arylamidase, naphthol-AS-BI phosphohydrolase, α-galactosidase, and β-glucosidase. Likewise, Trichosporon montevideense demonstrated esterase-lipase (C8), lipase (C14), valine arylamidase, naphthol-AS-BI phosphohydrolase, α-galactosidase, and β-glucosidase activity. T.coremiiforme and T. monteviidense isolated from turkeys were itraconazole resistant and amphotericin B, fluconazole, and voriconazole susceptible. Compared with human isolates, the MIC range and MIC values of turkey isolates to itraconazole were in a higher range limit in both species, while MIC values to amphotericin B, fluconazole, and voriconazole were in a lower range limit. Furthermore, the obtained ITS1—5.8rRNA—ITS2 fragment sequences were identical with T. coremiiforme and T. montevideense sequences isolated from humans indicating that these isolates are shared pathogens.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism

1999 ◽  
Vol 341 (1) ◽  
pp. 89-93 ◽  
Author(s):  
Gianluca TELL ◽  
Lucia PELLIZZARI ◽  
Gennaro ESPOSITO ◽  
Carlo PUCILLO ◽  
Paolo Emidio MACCHIA ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Congenital Hypothyroidism ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Structural Defects ◽  
Molecular Defect ◽  
Wild Type ◽  
Coding Region ◽  
Induced Fit ◽  
Helical Content

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in α-helical content upon interaction with DNA (‘induced fit’). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low α-helical content; however, in the Leu62Arg mutant, the gain in α-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

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