Biology subjects

Mathee, K.

Publications and source records attributed to Mathee, K..

3 recordsLinked to original sources

bioRxiv2018

Potential application of phage therapy for prophylactic treatment against Pseudomonas aeruginosa biofilm Infections.

The majority of the microbial activity in humans is in the form of biofilms, i.e., an exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotic treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. Antibiotic resistance has led to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ and in the presence of susceptible hosts can be used as natural, self-limiting, and profoundly penetrating antibacterial agents. The objective of this study is to identify active phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAO1 and the isogenic constitutively alginate-producing PDO300 strains. These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B, and P182). The analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance, but none of the susceptible strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the amount required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

microbiology↗

bioRxiv2018

Characterization of L-serine deaminases, SdaA (PA2448) and SdaB (PA5379), and their potential role in Pseudomonas aeruginosa pathogenesis

Regardless of the site of infectivity, all pathogens require high energetic influxes. This energy is required to counterattack the host immune system and in the absence the bacterial infections are easily cleared by the immune system. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. L-SD has been linked directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. The genome sequence analysis revealed the presence of two L-SD homologs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains of sdaA (PAOsdaA) and sdaB (PAOsdaB) with their isogenic parent P. aeruginosa PAO1. We demonstrate that P. aeruginosa is unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine. Both SdaA and SdaB contribute to L-serine deamination, 34 % and 66 %, respectively. Glycine was also shown to increase the L-SD activity especially from SdaB. Glycine-dependent induction requires the inducer serine. The L-SD activity from both SdaA and SdaB is inhibited by the amino acid L-leucine. These results suggest that P. aeruginosa L-SD is quite different from the characterized E. coli L-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as sole carbon source were isolated. In addition, suicide vectors were constructed which allow for selective mutation of the sdaA and sdaB genes on any P. aeruginosa strain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

microbiology↗

bioRxiv2018

Alginate-regulating genes are identified in the clinical cystic fibrosis isolate of Pseudomonas aeruginosa PA2192

Cystic fibrosis (CF) is a genetic disorder that leads to a buildup of mucus in the lungs ideal for bacterial colonization. When Pseudomonas aeruginosa enters the CF lung, it undergoes a conversion from nonmucoid to mucoid; colonization by a mucoid strain of P. aeruginosa greatly increases mortality. The mucoid phenotype is due to the production of alginate. The regulator of alginate production is the AlgT/U sigma factor. The observed phenotypic conversion is due to a mutation in the mucA gene coding for an anti-sigma factor, MucA, which sequesters AlgT/U. This mucoid phenotype is unstable when the strains are removed from the lung as they acquire second-site mutations. This in vitro reversion phenomenon is utilized to identify novel genes regulating alginate production. Previously, second-site mutations were mapped to algT/U, algO, and mucP, demonstrating their role in alginate regulation. Most of these studies were performed using a non-CF isolate. It was hypothesized that second site mutations in a clinical strain would be mapped to the same genes. In this study, a clinical, hyper-mucoid P. aeruginosa strain PA2192 was used to study the reversion phenomenon. This study found that PA2192 has a novel mucA mutation which was named them mucA180 allele. Twelve colonies were sub-cultured for two weeks without aeration at room temperature in order to obtain nonmucoid suppressors of alginate production (sap). Only 41 sap mutants were stable for more than 48 hours -- a reversion frequency of 3.9% as compared to ~90% in laboratory strains showing that PA2192 has a stable mucoid phenotype. This phenotype was restored in 28 of the 41 sap mutants when complemented with plasmids harboring algT/U. Four of the sap mutants are complemented by algO. Sequence analyses of the algT/U mutants have found no mutations in the coding region or promoter leading to the hypothesis that there is another, as yet unidentified mechanism of alginate regulation in this clinical strain.

microbiology↗