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

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.

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

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.

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy inPseudomonas aeruginosathrough Sigma Factor Competition

ABSTRACTMucoidy due to alginate overproduction by the Gram-negative bacteriumPseudomonas aeruginosafacilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption inde novosynthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoidP. aeruginosastrain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in thesemucA25mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (PalgD), but had no effect on PalgU, which controls transcription ofalgU. Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressingrpoNresulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for PalgD.IMPORTANCEChronic lung infections withP. aeruginosaare the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulatedP. aeruginosabacteria, leading to improved prognosis for patients with cystic fibrosis.