Genotypic Characterization of Pseudomonas aeruginosa Isolates from Eyes with Keratitis and Healthy Conjunctival Sacs

2021 ◽  
Author(s):  
Xiubin Ma ◽  
Qing Liu ◽  
Fangying Song ◽  
Yusen Huang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Group Versus ◽  
Therapeutic Interventions ◽  
Corneal Epithelial ◽  
Go Enrichment ◽  
Polymerase Chain ◽  
Upregulated Genes ◽  
Bacterial Secretion ◽  
Selection Of

Abstract Pseudomonas aeruginosa(P. aeruginosa) was a second most common commensal bacterium in healthy conjunctival sacs. When the corneal epithelial barrier is damaged, P. aeruginosa could cause keratitis, which progresses rapidly and results in corneal perforation and the loss of vision. However, the similarities and differences in characteristics between P. aeruginosa isolates from the eyes with keratitis and those from healthy conjunctival sacs are still poorly understood. In this study, four P. aeruginosa isolates from eyes with keratitis and three P. aeruginosa isolates from healthy conjunctival sacs were obtained, and genotypically characterized using Illumina high-throughput RNA sequencing. A total of 557 differentially expressed genes (DEGs) were found and included 332 upregulated genes and 225 downregulated genes in the keratitis group versus the healthy conjunctival sacs group. Of 557 DEGs, 11 DEGs analyzed with GO enrichment and the KEGG pathway were involved with the bacterial secretion system and pyoverdine metabolism and validated using quantitative reverse-transcription polymerase chain reaction. P. aeruginosa from eyes with keratitis induced more severe corneal infection and higher clinical scores in the mice. These results will contribute to develop alternative therapeutic interventions targeting virulence factors in these DEGs and facilitate the selection of therapeutic strategies.

scholarly journals Dietary fat clearance in type V hyperlipoproteinaemia secondary to a rare variant of human apolipoprotein E: the apolipoprotein E3 (Arg 136 → Ser)

2000 ◽  
Vol 83 (6) ◽  
pp. 615-622 ◽  
Author(s):  
Bernard Vialettes ◽  
Pascal Reynier ◽  
Catherine Atlan-Gepner ◽  
Nadia Mekki ◽  
Laurence Lesluyes-Mazzochi ◽  
...  
Keyword(s):  
Phenotypic Expression ◽  
Present Case Report ◽  
Human Apolipoprotein ◽  
Low Fat Diet ◽  
Mutant Variant ◽  
Restriction Polymorphism ◽  
Polymerase Chain ◽  
Type V ◽  
Selection Of

This present case report describes two siblings with severe type V hyperlipoproteinaemia, diagnosed very early in life and due to the combination of the common apolipoprotein (Apo) E2 allele and a rare mutant variant of ApoE, ApoE3 (Arg 136 → Ser). Phenotyping of ApoE falsely identified E2/E2 phenotype. The presence of mutated ApoE was suspected on an unusual restriction polymorphism of a Hha 1 restriction site and confirmed by sequence analysis of the cloned polymerase chain reaction fragment of exon 4 and familial segregation study. The severity of the hypertriacylglycerolaemia was modulated by the lipid content of the diet. A low-fat diet enriched in medium-chain triacylglycerol (TAG) decreased but did not normalize plasma TAG levels in both affected patients of the pedigree. A standardized lipid-enriched test meal showed a marked impairment of TAG-rich lipoprotein (TRL) clearance, especially the exogeneous TRL bearing ApoB-48 which still represented 79 % of total TRL 7 h after the fat load. Finally, differences between the male and female siblings with the existence of a consanguine relationship in their parents suggested the involvement of other genetic factors in modulating the severity of phenotypic expression. This observation reinforces the usefulness of genotyping of ApoE for the characterization of genetic hypertriacylglycerolaemia and selection of the appropriate diet and treatment.

The use of deficiencies to determine essential gene content in the let-56–unc-22 region of Caenorhabditis elegans

Genome ◽  
1993 ◽  
Vol 36 (6) ◽  
pp. 1148-1156 ◽  
Author(s):  
Jacquie E. Schein ◽  
Marco A. Marra ◽  
Guy M. Benian ◽  
Chris Fields ◽  
David L. Baillie
Keyword(s):  
Polymerase Chain Reaction ◽  
Caenorhabditis Elegans ◽  
Genomic Sequence ◽  
Essential Gene ◽  
Chain Reaction ◽  
Radiation Induced ◽  
Practical Tool ◽  
Polymerase Chain ◽  
Selection Of

We have investigated the possibility of using the polymerase chain reaction to detect deletions of coding elements in the unc-22–let-56 interval on chromosome IV in the nematode Caenorhabditis elegans. Our analysis of approximately 13 kb of genomic sequence immediately to the left of the unc-22 gene resulted in the identification of four possible genes. Partial cDNAs have been identified for three of them. To determine whether any of these coding elements are essential for development, we required a method for the induction and selection of mutations in these elements. Our approach was to identify a set of formaldehyde and gamma radiation induced unc-22 mutations that mapped to the unc-22–let-56 region, and then employ polymerase chain reaction methodology to identify deficiencies that affected one or more of the four identified coding elements. Two small deficiencies were identified in this manner. Characterization of these deficiencies shows that there are no coding elements between unc-22 and let-56 (the nearest mutationally identified gene to the left of unc-22), which are required in development under laboratory conditions. We conclude that the polymerase chain reaction is a practical tool for the detection of deletions of coding elements identified in this region, and that characterization of such deficiencies provides a method for assessing whether or not these elements are required for development.Key words: Caenorhabditis elegans, deficiencies, coding elements, unc-22.

scholarly journals Azithromycin in Pseudomonas aeruginosa Biofilms: Bactericidal Activity and Selection of nfxB Mutants

2009 ◽  
Vol 53 (4) ◽  
pp. 1552-1560 ◽  
Author(s):  
Xavier Mulet ◽  
María D. Maciá ◽  
Ana Mena ◽  
Carlos Juan ◽  
José L. Pérez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bactericidal Activity ◽  
Cross Resistance ◽  
Resistance Development ◽  
Viable Cells ◽  
Knockout Mutants ◽  
Lung Infections ◽  
Selection Of

ABSTRACT Azithromycin (AZM) has shown promising results in the treatment of Pseudomonas aeruginosa chronic lung infections such as those occurring in cystic fibrosis (CF) patients. We evaluated the effect of hypermutation and alginate hyperproduction on the bactericidal activity and resistance development to AZM in P. aeruginosa biofilms. Strains PAO1, its mucA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were used. Biofilms were incubated with several AZM concentrations for 1, 2, 4, or 7 days, and the numbers of viable cells were determined. During the first 2 days, AZM showed bactericidal activity for all the strains, but in extended AZM incubation for strain PAOMS and especially strain PAOMSA, a marked increased in the number of viable cells was observed, particularly at 4 μg/ml. Biofilms formed by the lineages recovered from the 7-day experiments showed enhanced AZM resistance. Furthermore, most of the independent lineages studied, including those obtained from biofilms treated with AZM concentrations as low as 0.5 μg/ml, showed MexCD-OprJ hyperexpression and mutations in nfxB. The role of nfxB mutation in AZM resistance was further confirmed through the characterization of nfxB and mexD knockout mutants. Results from this work show that, although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected and that, furthermore, they frequently show cross-resistance to other unrelated antipseudomonal agents such as ciprofloxacin or cefepime but hypersusceptibility to others such as imipenem or tobramycin. Therefore, these results should help guide the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.

scholarly journals Extended-Spectrum Cephalosporinases in Pseudomonas aeruginosa

2009 ◽  
Vol 53 (5) ◽  
pp. 1766-1771 ◽  
Author(s):  
José-Manuel Rodríguez-Martínez ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
In Vitro Selection ◽  
Resistance Mechanism ◽  
Cloning And Sequencing ◽  
Alanine Residue ◽  
Resistant Strains ◽  
Imipenem Resistance ◽  
Selection Of

ABSTRACT The characterization of AmpC-type β-lactamases was performed in a collection of 32 clinical Pseudomonas aeruginosa isolates with intermediate susceptibility or resistance to imipenem and ceftazidime. Twenty-one out of those 32 isolates overexpressed AmpC β-lactamase, and the MICs of ceftazidime and imipenem were reduced after cloxacillin addition. Cloning and sequencing identified 10 AmpC β-lactamase variants. Reduced susceptibility to imipenem, ceftazidime, and cefepime was observed only with recombinant P. aeruginosa strains expressing an AmpC β-lactamase that had an alanine residue at position 105. The catalytic efficiencies (k cat/Km ) of the AmpC variants possessing this residue were increased against oxyiminocephalosporins and imipenem. In addition, we show here that those AmpC variants constitute a favorable background for the in vitro selection of imipenem-resistant strains. This report identified a novel resistance mechanism that may contribute to imipenem resistance in P. aeruginosa.

Characterization of tetracycline resistance genes in Escherichia coli isolated from feedlot cattle administered therapeutic or subtherapeutic levels of tetracycline

2013 ◽  
Vol 59 (4) ◽  
pp. 287-290 ◽  
Author(s):  
T.W. Alexander ◽  
X. Jin ◽  
Q. Li ◽  
S. Cook ◽  
T.A. McAllister
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Feedlot Cattle ◽  
E Coli ◽  
Treatment Regimes ◽  
Tetracycline Resistance Genes ◽  
Polymerase Chain ◽  
Selection Of

The effect of administering feedlot cattle subtherapeutic levels of chlortetracycline (CT) or CT and therapeutic levels of oxytetracycline (CT-OX) on resistance genotypes in Escherichia coli was investigated. Detection of genes tet(A), tet(B), and tet(C) encoded by tetracycline-resistant isolates (CT, N = 77; CT-OX, N = 99) was performed by multiplex polymerase chain reaction (PCR). Prevalence of tet(A) was similar in isolates across treatment regimes; however, prevalence of tet(B) was lower (18% versus 34%; P < 0.05) and tet(C) was higher (46% versus 28%; P < 0.05) in CT isolates compared with CT-OX isolates. To further characterize selection of resistance genotypes in E. coli, a group of intermediately tetracycline-resistant E. coli (N = 48) was analyzed. The tet(C) gene was present in 92% of these isolates. Copies of tet(C) transcripts, analyzed by real-time PCR, indicated that upregulation did not occur in tetracycline-resistant isolates when compared with intermediately resistant isolates. The minimum inhibitory concentrations of tetracycline, chlortetracycline, and oxytetracycline were also tested on isolates with different resistance genes. The minimum inhibitory concentration was dependent on the tetracycline analogue and the nature of encoded resistance. These data indicate that tetracycline analogues should not be used interchangeably to evaluate resistance and that prevalence of resistance genes in E. coli can vary according to the tetracycline analogue administered to cattle.

Applications of Transmission Electron Microscopy in the Characterization of Metal Machinability

1968 ◽  
Vol 26 ◽  
pp. 442-443 ◽  
Author(s):  
L.E. Murr ◽  
A.B. Draper
Keyword(s):  
Electron Microscopy ◽  
State Physics ◽  
Test Material ◽  
Milling Machine ◽  
Rotary Table ◽  
Solid State Physics ◽  
Materials Properties ◽  
Transmission Electron ◽  
Selection Of

The industrial characterization of the machinability of metals and alloys has always been a very arbitrarily defined property, subject to the selection of various reference or test materials; and the adoption of rather naive and misleading interpretations and standards. However, it seems reasonable to assume that with the present state of knowledge of materials properties, and the current theories of solid state physics, more basic guidelines for machinability characterization might be established on the basis of the residual machined microstructures. This approach was originally pursued by Draper; and our presentation here will simply reflect an exposition and extension of this research.The technique consists initially in the production of machined chips of a desired test material on a horizontal milling machine with the workpiece (specimen) mounted on a rotary table vice. A single cut of a specified depth is taken from the workpiece (0.25 in. wide) each at a new tool location.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

The Study of Bacillus Subtils Antimicrobial Activity on Some of the Pathological Isolates

2019 ◽  
Vol 9 (02) ◽  
Author(s):  
Hussein A Kadhum ◽  
Thualfakar H Hasan2
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Bacterial Growth ◽  
Broad Spectrum ◽  
Inhibitory Activity ◽  
Bacterial Pathogens ◽  
Inhibitory Ability ◽  
Selection Of

The study involved the selection of two isolates from Bacillus subtilis to investigate their inhibitory activity against some bacterial pathogens. B sub-bacteria were found to have a broad spectrum against test bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. They were about 23-30 mm and less against Klebsiella sp. The sensitivity of some antibodies was tested on the test samples. The results showed that the inhibitory ability of bacterial growth in the test samples using B. subtilis extract was more effective than the antibiotics used.

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