New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity.

BACKGROUND: Helicobacter pylori is a fastidious pathogen that is required a complicated medium for growth. Invading epithelial cells of the stomach. H. pylori virulence factors are classified by function, acidic resistivity, adhesion, chemotaxis and motility, molecular mimicry, immunological invasion and modulation, and toxins formation such as cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA). This study aims to determine a simple and innovative technique to isolate H. pylori from gastric biopsies and assess pathogenicity by virulence factor gene detection. METHODS: A total of 200 patients who were suspected of having H. pylori infection had two antral gastric biopsies undertaken. A rapid urease test (RUT) was used for one, and Brain Heart Infusion broth (BHI) was used to cultivate the other. The molecular study included diagnostics utilizing the 16sRNA housekeeping gene along with the identification of the virulence factors genes (cagA, cagT, and vacA) and sequencing, RESULT: Of the 200 antral gastric biopsies collected, 135 were positive rapid urease tests, and 17 H. pylori isolates were successfully obtained from 135 biopsies. The 16SrRNA as a housekeeping gene is confirmed, and about 53%, 70.5%, and 82.3% of the 17 isolates show carrying cagA, cagT, and vacA genes, respectively. All peptic ulcer isolates have the cagA gene, while Gastroesophageal Reflux Disease (GERD) and non-peptic ulcer disease (NPUD) isolates show the lack of the cagA gene. All bacteria, which were isolated from peptic ulcer, nodular gastritis, and gastritis patients, have a vacA gene. CONCLUSION: The effective method for isolating H. pylori is centrifuging the transport broth after 24 h of incubation. The cagA toxin causes peptic ulcer while vacA toxin induces several histopathological changes in the stomach. Three virulence genes were present in all peptic ulcer-causing bacteria, while only one or none were present in the GERD and NPUD biopsy isolates.

The secrets of environmental Pseudomonas aeruginosa in slaughterhouses: Antibiogram profile, virulence, and antibiotic resistance genes.

Environmental pollution is a serious problem that can cause sicknesses, fatality, and biological contaminants such as bacteria, which can trigger allergic reactions and infectious illnesses. There is also evidence that environmental pollutants can have an impact on the gut microbiome and contribute to the development of various mental health and metabolic disorders. This study aimed to study the antibiotic resistance and virulence potential of environmental Pseudomonas aeruginosa (P. aeruginosa) isolates in slaughterhouses. A total of 100 samples were collected from different slaughterhouse tools. The samples were identified by cultural and biochemical tests and confirmed by the VITEK 2 system. P. aeruginosa isolates were further confirmed by CHROMagar™ Pseudomonas and genetically by rpsL gene analysis. Molecular screening of virulence genes (fimH, papC, lasB, rhlI, lasI, csgA, toxA, and hly) and antibiotic resistance genes (blaCTX-M, blaAmpC, blaSHV, blaNDM, IMP-1, aac(6')-Ib-, ant(4')IIb, mexY, TEM, tetA, and qnrB) by PCR and testing the antibiotic sensitivity, biofilm formation, and production of pigments, and hemolysin were carried out in all isolated strains. A total of 62 isolates were identified as P. aeruginosa. All P. aeruginosa isolates were multidrug-resistant and most of them have multiple resistant genes. blaCTX-M gene was detected in all strains; 23 (37.1%) strains have the ability for biofilm formation, 33 strains had virulence genes, and 26 isolates from them have more than one virulence genes. There should be probably 60 (96.8%) P. aeruginosa strains that produce pyocyanin pigment. Slaughterhouse tools are sources for multidrug-resistant and virulent pathogenic microorganisms which are a serious health problem. Low-hygienic slaughterhouses could be a reservoir for resistance and virulence genes which could then be transferred to other pathogens.

Physical-chemical properties and acellular bioactivity of newly prepared nano-tricalcium silicate-58s bioactive glass-based endodontic sealer.

OBJECTIVES: To evaluate the physiochemical properties and apatite-forming ability of a newly prepared nano-tricalcium silicate-58s bioactive glass-based endodontic sealer (C3S-BG-P) and compare its results with the Nishika BG canal sealer and BioRoot™ RCS. METHODS: The physicochemical properties (setting time, flow, solubility, film thickness, and radiopacity) of C3S-BG-P, Nishika BG canal sealer, and BioRoot™ RCS were evaluated in accordance with ANSI/ADA 57/2000 (reaffirmed 2012) and ISO 6876:2012 for root canal sealing materials. The in vitro apatite-forming ability was evaluated after 28 days of immersion of disc-shaped specimens in phosphate-buffered saline (PBS) using field emission scanning electron microscopy and energy-dispersive X-ray spectroscopy. RESULTS: The results of physiochemical tests indicated that all the tested sealers complied with the ADA and ISO standards; however, the solubility of the BioRoot did not meet the two standards. C3S-BG-P revealed significantly superior properties in all physicochemical tests compared to Nishika and BioRoot; however, the solubility of Nishika was significantly lower than that of C3S-BG-P. Furthermore, all tested sealers exhibited apatite precipitation on their surfaces after 28 days of immersion in PBS. CONCLUSIONS: C3S-BG-P had superior physicochemical properties, which mitigated the disadvantages of calcium silicate-based sealers. Moreover, it exhibited apatite precipitation after immersion in PBS. Further in vivo studies utilizing animal models or clinical studies are necessary to support the rationale of the newly developed sealer for clinical application.

Purification and characterization of catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) using the local isolate of phenol-degrading Pseudomonas putida.

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.

Remineralization ability of different root canal sealers.

Aims: This research was designed to contrast the biocompatibility and remineralization ability of different sealers (BioRoot, MTA-FillApex and GuttaFlow-Bioseal). Method: Twenty rabbits were used in this study, they were randomly divided into 4 groups equally depending on the observation time"3,7,14, and 28 days" post-implantation. Each rabbit was generally anesthetized,"7cm"long incision was made on the skin of the right and left sides of the ventral aspect of the mandible of each rabbit, 4 bony cavities of approximately"5mm"in depth and"2mm"in diameter (2 cavities on the left side and 2 cavities on the right side of mandible of each rabbit) were made in the cortical surface of the buccal alveolar bone. The sealers mixed depend on manufactural instructions and immediately insert into the prepared cavities (in the right side the BioRoot and MTA-FillApex were placed while on the left side, GuttaFlow-Bioseal was placed in one cavity and the other cavity was left unfilled as control).The same volume of each sealer was placed in the corresponding cavity using disposable syringes. After each observation period, the animals were sacrificed and bone biopsy from the tested area was taken and examined histologically using Olympus light microscopy at"400X"magnification. Results: The obtained data were analyzed through non-parametric statistical tests using SPSS software version"22".Kruskal-Wallis test and Mann - Whitney test were utilized at"0.05"levels of significance to evaluate the results. GuttaFlow-Bioseal displayed excellent biocompatibility in comparison to other groups indicated by low inflammatory tissue reaction at all evaluation intervals. While the BioRoot group represented better osteo-conductivity although statistically not significant than GuttaFlow-Bioseal group. Conclusion: BioRoot and GuttaFlow-Bioseal showed higher osteo-conductivity and biocompatibility than MTA-FillApex. However, all sealer used in this study were well tolerated by bone tissue and might accelerate bone repair.

Effect of Oral Vitamin D3 on Dental Caries: An In-Vivo and In-Vitro Study.

Aim Vitamin D3 plays an important role in affecting the overall remineralization process of the dentition. The use of supplements help to keep the levels at optimum and thus reduce the chances of treating very early lesion of caries. Hence the aim was to investigate the indirect effects of oral vitamin D3 on microhardness and elemental weight percentage of Calcium (Ca) and Phosphorous (P) in enamel surface with an artificially initiated carious lesion. Methods The 120 extracted premolars were randomly divided into five groups according to salivary immersion. Each group had a total of twenty-four participants, with the following characteristics: control +ve: sound enamel; control -ve: only subjected to pH cycle; A: pH cycle and immersion in control saliva; B: pH cycle and saliva collected after three weeks; and C: pH cycle and saliva collected after six weeks. The unstimulated saliva was collected from (40) adult volunteers receiving vitamin D3 1000IU gel capsules daily for six weeks. Before each vitamin D3 intake, 10 mL of unstimulated control saliva was collected from each participant. Then other 10 ml. were collected after three and six weeks of vitamin D receiving periods. Saliva immersion time (12 hours). Enamel surface was assessed by Vicker's Microhardness machine and (X-ray fluorescence - XRF) spectrometer. Results For all specimens, there was a significant decrease in both (Ca and P weight %) after demineralization and then they significantly increased after receiving vitamin D3. The microhardness and elemental analysis provide confirmed results that were represented as a statistically significant difference at (P≤ 0.05) between groups that received vitamin D3 and those without vitamin D3 dosage. Conclusions Oral vitamin D3 has a significant potential in motivating remineralization of early lesions on the enamel surfaces representing improved surface microhardness and minerals content (Ca and P weight %) of demineralized tooth surfaces.

A Novel Genetic Determination of a Lectin Gene in Iraqi Acinetobacter baumannii Isolates and Use of Purified Lectin as an Antibiofilm Agent.

BACKGROUND: Lectin was initially called hemagglutinin or agglutinin because of its capacity to agglutinate human as well as human erythrocytes. They are a heterogeneous group of proteins or glycoproteins of nonimmune origin. Because of their chemical properties, they have become a useful tool in several fields such as immunology, cell biology, molecular biology, membrane structure, pharmacology, cancer research, clinical chemistry, and genetic engineering. OBJECTIVE: The wide applications of lectins users urged the need to isolate lectins from a new strain of bacteria can produce new and high yield of lectin because the current production of lectin from Pseudomonas spp. is very expensive. The goal of this study was to screen the ability of Acinetobacter baumannii isolates to produce lectin and detection of its phenotypic and genotypic profiles and detection of lectin ability to inhibit ofbiofilm formation. METHODS: Fifty-one isolates from different sources were collected and detected genetically by using the recA gene. Phenotypic detection of lectin by using semi-quantitative analysis and quantitative analysis in microtiter plate. Genotypic detection of lectin by designed lec gene and used PCR technique. The lectin was extracted by using glass beads and purified by chromatographyic technique followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for determination the molecular size of lectin and finally detection the spectrum of biofilm inhibition by the purified lectin toward biofilm producers. RESULTS: Of 51 A. baumannii isolates, 17 (33.3%) have been found to produce lectin. Ten of 17 were sequenced, of which 2 were submitted and tested by the gene bank National Center for Biotechnology Information (NCBI), and accession numbers (KX766405.1 and KX766406.1) were obtained. These 17 isolates were phenotypically and genotypically positive for lectin and showed different lec gene expression in semi-quantitative and quantitative analysis. The activities ranged between 4-128 U/mL. Lectin purified by ammonium sulfate precipitation was used to inhibit biofilm formation. We found reduction at three different types of bacteria ranging from 26% for Klebsiella pneumonia, 46.7% for P. stutzeri and 53% for A. baumannii. These results suggested that lectin has a promising application as an antibiofilm agent to combat the growing number of multidrug-resistant pathogen-associated infections. CONCLUSIONS: Lectin has been detected recently in A. baumannii, but the genetic property of this lectin has not yet been fully studied. In our study, we determined the presence of the lectin gene (lec gene) in A. baumannii by using PCR technique, and lec PCR products were identified with various source of isolation and sequenced to screening for epidemiology and submitted to the gene bank NCBI under accession number (KX766405.1 and KX766406.1). HIGHLIGHTS: A. baumannii has an ability to produce lectin protein; Lec gene was detected in A. baumannii, and the sequence was recorded under accession number KX766405.1 and KX766406.1.; Lectin was extracted by glass beads and purified by chromatographyic technique; Lectin had strong effect against biofilm formation.

Identification of a novel function for the FtsL cell division protein from Escherichia coli K12.

Analysis of the essential cell division protein FtsL demonstrates the partial conservation of a cysteine-pair within the trans-membrane region which itself is flanked by histidine-pairs in the cytosol and periplasm. Similar arrangements of such amino acids are seen in proteins known to transport/bind metal ions in biological systems. Heterologous expression of ftsL in Escherichia coli K12 confers a Zn(II)-sensitive phenotype and alteration of the candidate metal-ion binding residues cysteine or histidine substantially alters this phenotype. Whilst the cysteine/histidine replacement derivatives of ftsL were able to complement an otherwise ftsL-null strain, the derivative carrying ftsL lacking the cysteine pair was sensitive to raised metal-ion concentrations in the media. We show that ftsL can confer a metal-ion sensitive phenotype and that trans-membrane cysteine residues play a role in FtsL function in elevated metal-ion concentrations.