scholarly journals Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and its Prediction of Resistance Development Through In-silico Transcriptomic Analysis

2021 ◽  
Author(s):  
Abishek Kumar B ◽  
Bency Thankappan ◽  
Angayarkanni Jayaraman ◽  
Akshita Gupta
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Gene Expressions ◽  
Resistance Development ◽  
Adaptive Resistance ◽  
Antibiotic Response ◽  
Secretory System ◽  
Kinetics Of ◽  
Alginate Biosynthesis

Pseudomonas aeruginosa causes severe life-threatening infections and are difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance, which prevents the entry of antibiotics into cytosol of the cell. Among different groups of antibiotics, aminoglycosides show superior antibiotic response and are used as a parental antibiotic for treatment. This study aims to determine the kinetics of adaptive resistance development and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. In vitro antibiotic exposure to P. aeruginosa was performed and optical density of the cells were monitored for every 12 hours until 72 hours. The growth pattern plotted in graph represents the kinetics of adaptive resistance developed to respective antibiotics. The transcriptomic profile of P. aeruginosa PA14 to post exposed antibiotic was taken from Gene Expression Omnibus (GEO), NCBI. The gene expressions of two datasets were analyzed by case-control study. Tobramycin exposed P. aeruginosa failed to develop adaptive resistance in 0.5ug/mL, 1ug/mL and 1.5ug/mL of its MIC. Whereas, amikacin and gentamicin treated P. aeruginosa developed tolerance in the inhibitory concentrations of the antibiotics. This depicts the superior in vitro response of tobramycin over the gentamicin and amikacin. Furthermore, tobramycin treated P. aeruginosa microarray analysis resulted in low expression of catalytic enzyme 16s rRNA Methyltransferase E, B & L, alginate biosynthesis genes and several proteins of Type 2 Secretory System (T2SS) and Type 3 Secretory System (T3SS). The Differentially Expressed Genes (DEGs) of alginate biosynthesis, and RNA Methyltransferases suggests increased antibiotic response and low probability of developing resistance. The use of tobramycin as a parental antibiotic with its synergistic combination might combat P. aeruginosa with increased response.

scholarly journals Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and Its Predicted Gene Regulations through In-Silico Transcriptomic Analysis

2021 ◽  
Vol 12 (3) ◽  
pp. 630-645
Author(s):  
Abishek Kumar Kumar B. ◽  
Bency Thankappan ◽  
Angayarkanni Jayaraman ◽  
Akshita Gupta
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
In Silico ◽  
Secretion System ◽  
Transcriptomic Analysis ◽  
Antibiotic Tolerance ◽  
Parenteral Antibiotic ◽  
Antibiotic Response ◽  
Kinetics Of ◽  
Alginate Biosynthesis

Pseudomonas aeruginosa causes chronic infections, such as cystic fibrosis, endocarditis, bacteremia, and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance mechanism, which prevents the entry of antibiotics into the cytosol of the cell to achieve tolerance. Among the different groups of antibiotics, aminoglycosides are used as a parenteral antibiotic for the treatment of P. aeruginosa. This study aimed to determine the kinetics of antibiotic tolerance and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed to P. aeruginosa at their MICs and the experimental setup was monitored for 72 h, followed by the measurement of optical density every 12 h. The growth of P. aeruginosa in the MICs of antibiotics represented the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. The transcriptomic profile of antibiotic exposed P. aeruginosa PA14 was taken from the Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin-exposed P. aeruginosa failed to develop tolerance in MICs of 0.5 µg/mL, 1 µg/mL, and 1.5 µg/mL, whereas amikacin- and gentamicin-treated P. aeruginosa developed tolerance. This illustrated the superior in vitro response of tobramycin over gentamicin and amikacin. Further, in silico transcriptomic analysis of tobramycin-treated P. aeruginosa resulted in differentially expressed genes (DEGs), enriched in 16s rRNA methyltransferase E, B, and L, alginate biosynthesis genes, and several proteins of the type II secretion system (T2SS) and type III secretion system (T3SS). The regulation of mucA in alginate biosynthesis, and gidB in RNA methyltransferases, suggested an increased antibiotic response and a low probability of developing resistance during tobramycin treatment. The use of tobramycin as a parenteral antibiotic with its synergistic combination might combat P. aeruginosa with increased response.

scholarly journals Pseudomonas aeruginosa PAO 1 In Vitro Time–Kill Kinetics Using Single Phages and Phage Formulations—Modulating Death, Adaptation, and Resistance

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 877
Author(s):  
Ana Mafalda Pinto ◽  
Alberta Faustino ◽  
Lorenzo M. Pastrana ◽  
Manuel Bañobre-López ◽  
Sanna Sillankorva
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phage Therapy ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Chronic Infections ◽  
Swarming Motility ◽  
Resistance Development ◽  
Healthcare Settings ◽  
Time Kill

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

scholarly journals Development of Resistance in Wild-Type and Hypermutable Pseudomonas aeruginosa Strains Exposed to Clinical Pharmacokinetic Profiles of Meropenem and Ceftazidime Simulated In Vitro

2007 ◽  
Vol 51 (10) ◽  
pp. 3642-3649 ◽  
Author(s):  
Beate Henrichfreise ◽  
Irith Wiegand ◽  
Ingeborg Luhmer-Becker ◽  
Bernd Wiedemann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Parent Strain ◽  
Resistance Mechanisms ◽  
In Vitro Models ◽  
Wild Type ◽  
Resistance Development ◽  
Development Of Resistance ◽  
Pharmacokinetic Profiles ◽  
Mutant Prevention Concentration

ABSTRACT In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.

scholarly journals Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 123-134 ◽  
Author(s):  
Grazieli Marinheiro Machado ◽  
Ester Siqueira Caixeta ◽  
Carolina Madeira Lucci ◽  
Rodolfo Rumpf ◽  
Maurício Machaim Franco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Morphological Characteristics ◽  
Tunnel Construction ◽  
Agarose Gel ◽  
Male And Female ◽  
Embryo Size ◽  
And Gender ◽  
Phosphate Buffered Saline ◽  
Kinetics Of

SummaryThe objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Liudmila Zakharova ◽  
Hikmet Nural ◽  
Mohamed A Gaballa
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Smooth Muscle Actin ◽  
Fold Increase ◽  
Gene Expressions ◽  
Mesenchymal Transition ◽  
Cell Outgrowth ◽  
Pluripotency Genes

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.

scholarly journals Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model.

1997 ◽  
Vol 41 (4) ◽  
pp. 823-826 ◽  
Author(s):  
Y Q Xiong ◽  
J Caillon ◽  
M F Kergueris ◽  
H Drugeon ◽  
D Baron ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Single Dose ◽  
Ex Vivo ◽  
Day Treatment ◽  
Total Daily Dose ◽  
Once Daily ◽  
Adaptive Resistance ◽  
Dosing Regimens

Adaptive resistance following the first exposure to aminoglycosides is a recently described in vitro phenomenon in Pseudomonas aeruginosa and other aerobic gram-negative bacilli. We investigated the in vivo relevance of adaptive resistance in P. aeruginosa following a single dose of amikacin in the experimental rabbit endocarditis model. Rabbits with P. aeruginosa endocarditis received either no therapy (control) or a single intravenous (i.v.) dose of amikacin (80 mg/kg of body weight) at 24 h postinfection, after which they were sacrificed at 5, 8, 12, 16, or 24 h postdose. Excised aortic vegetations were subsequently exposed ex vivo to amikacin at 2.5, 5, 10 or 20 times the MIC for 90 min. In vivo adaptive resistance was identified when amikacin-induced pseudomonal killing within excised aortic vegetations was less in animals receiving single-dose amikacin in vivo than in vegetations from control animals not receiving amikacin in vivo. Maximal adaptive resistance occurred between 8 and 16 h after the in vivo amikacin dose, with complete refractoriness to ex vivo killing by amikacin seen at 12 h postdose. By 24 h postdose, bacteria within excised vegetations had partially recovered their initial amikacin susceptibility. In a parallel treatment study, we demonstrated that amikacin given once daily (but not twice daily) at a total dose of 80 mg/kg i.v. for 1-day treatment significantly reduced pseudomonal densities within aortic vegetations versus those in untreated controls. When therapy was continued for 3 days with the same total daily dose (80 mg/kg/day), amikacin given once or twice daily significantly reduced intravegetation pseudomonal densities versus those in controls. However, amikacin given once daily was still more effective than the twice-daily regimen. These data confirm the induction of aminoglycoside adaptive resistance in vivo and further support the advantages of once-daily aminoglycoside dosing regimens in the treatment of serious pseudomonal infections.

scholarly journals Imipenem/Cilastatin/Relebactam Alone and in Combination against Pseudomonas aeruginosa in the In Vitro Pharmacodynamic Model

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Iris H. Chen ◽  
David P. Nicolau ◽  
Joseph L. Kuti
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Plasma Concentrations ◽  
Multidrug Resistant ◽  
Pharmacodynamic Model ◽  
Resistance Development ◽  
Content Type ◽  
Combination Studies ◽  
The Mean ◽  
Combined Drugs

ABSTRACT Combination therapy may enhance imipenem/cilastatin/relebactam’s (I/R) activity against Pseudomonas aeruginosa and suppress resistance development. Human-simulated unbound plasma concentrations of I/R at 1.25 g every 6 h (h), colistin at 360 mg daily, and amikacin at 25 mg/kg daily were reproduced alone and in combination against six imipenem-nonsusceptible P. aeruginosa isolates in an in vitro pharmacodynamic model over 24 h. For I/R alone, the mean reductions in CFU ± the standard errors by 24 h were −2.52 ± 0.49, −1.49 ± 0.49, −1.15 ± 0.67, and −0.61 ± 0.10 log10 CFU/ml against isolates with MICs of 1/4, 2/4, 4/4, and 8/4 μg/ml, respectively. Amikacin alone also resulted in 24 h CFU reductions consistent with its MIC, while colistin CFU reductions did not differ. Resistant subpopulations were observed after 24 h in 1, 4, and 3 I/R-, colistin-, and amikacin-exposed isolates, respectively. The combination of I/R and colistin resulted in synergistic (n = 1) or additive (n = 2) interactions against three isolates with 24-h CFU reductions ranging from −2.62 to −4.67 log10 CFU/ml. The combination of I/R and amikacin exhibited indifferent interactions against all isolates, with combined drugs achieving −0.51- to −3.33-log10 CFU/ml reductions. No resistant subpopulations were observed during I/R and colistin combination studies, and when added to amikacin, I/R prevented the emergence of amikacin resistance. Against these six multidrug-resistant P. aeruginosa, I/R alone achieved significant CFU reductions against I/R-susceptible isolates. Combinations of I/R plus colistin resulted in additivity or synergy against some P. aeruginosa, whereas the addition of amikacin did not provide further antibacterial efficacy against these isolates.

scholarly journals Comparative Survival and the Cold-Induced Gene Expression of Pathogenic and Nonpathogenic Vibrio Parahaemolyticus from Tropical Eastern Oysters during Cold Storage

2020 ◽  
Vol 17 (6) ◽  
pp. 1836
Author(s):  
Francisco Alarcón Elvira ◽  
Violeta T. Pardío Sedas ◽  
David Martínez Herrera ◽  
Rodolfo Quintana Castro ◽  
Rosa María Oliart Ros ◽  
...  
Keyword(s):  
Low Temperatures ◽  
Vibrio Parahaemolyticus ◽  
Cold Shock ◽  
Gene Expressions ◽  
Reverse Transcription Pcr ◽  
Naturally Occurring ◽  
Gene Response ◽  
Eastern Oysters ◽  
Kinetics Of

Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by −0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.

Bidirectional Oscillatory Shear Stress Increases Pro-Atherogenic Gene Expressions (I-CAM1, E-Selectin and IL-6) in Endothelial Cells

2011 ◽  
Author(s):  
Amlan Chakraborty ◽  
Venkatakrishna R. Jala ◽  
Sutirtha Chakraborty ◽  
R. Eric Berson ◽  
M. Keith Sharp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Atherosclerotic Plaque ◽  
Inflammatory Diseases ◽  
Leukotriene B4 ◽  
Oscillatory Shear ◽  
Gene Expressions

Wall shear stress (WSS) plays a key role in altering intracellular pathways and gene expression of endothelial cells, and has significant impacts on atherosclerotic plaque development (1–3). Further, the atherogenic regulators Leukotriene B4 (LTB4) and Lipopolysaccharide (LPS) have significant impacts on the pathophysiology of many inflammatory diseases. This study investigates the effects of oscillatory shear directionality on pro-atherogenic gene expression (I-CAM, E-Selectin, and IL-6) in the presence of LTB4 and LPS. An orbital shaker was used to expose the endothelial cells to oscillatory shear in culture dishes, and Computational fluid dynamics (CFD) was applied to quantify the shear stress on the bottom of the orbiting dish. Directionality of oscillatory shear was characterized by a newly developed hemodynamic parameter — Directional oscillatory shear index (DOSI), which was demonstrated in a previous study to significantly impact cell morphology (4). Results showed that DOSI significantly altered gene expression. Therefore, directionality of shear modulates atherosclerotic gene expression in vitro and thus, may influence the formation of atherosclerotic plaque in vivo.

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