Serine protease-dependent degradation of IL-6 by Pseudomonas aeruginosa in an in vitro model of bacterial-viral coinfection

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

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Hydrolysis of cefazolin by enzymes produced by Pseudomonas aeruginosa after exposure to ceftazidime in vitro

Vojnosanitetski pregled ◽

10.2298/vsp0910785p ◽

2009 ◽

Vol 66 (10) ◽

pp. 785-790

Author(s):

Paraskevi Papaioannidou ◽

Vassilios Nitsas ◽

Vassiliki Mirtsou-Fidani

Keyword(s):

Pseudomonas Aeruginosa ◽

In Vitro Model ◽

Short Exposure ◽

Test Results ◽

Diffusion Test ◽

High Tendency ◽

Resistant Strains ◽

Vitro Model ◽

Hydrolysis Of

Background/Aim. Sometimes resistance of Pseudomonas aeruginosa (Ps. aeruginosa) is developed during antibiotic treatment, in spite of the initial susceptibility in vitro. The aim of this study was to use an in vitro model for the study of the development of resistant strains of Ps. aeruginosa after a short exposure to ceftazidime, and to study the hydrolysing capacity of ?-lactamases produced by the resistant strains. Methods. Among 563 clinical strains of Ps. aeruginosa, 37 multisensitive strains were collected for the study. After being identified, strains with simultaneous sensitivity to 5 expanded spectrum cephalosporins were chosen. For each strain, the minimal inhibitory concentration (MIC) of the 5 expanded spectrum cephalosporins was determined, and the production of extended spectrum ?-lactamases (ESBL) was excluded by the double-disc synergy diffusion test. Strains non producing ESBL were cultivated in concentrations of ceftazidime equal to MIC?2 and MIC?4. After 24 hours of culture, the development of resistant strains was estimated and the cephalosporinase activity of the produced ?-lactamases was determined by their ability to hydrolyse cefazolin. Hydrolysis of cefazolin was studied by measuring the change of its absorbance on 272 nm using a Shimadzu 160A spectrophotometer. The hydrolyzing capacity of the enzymes was expressed as the percentage of the antibiotic, which was hydrolysed in 10 sec. Results. A total of 60% and 50% of strains developed resistant strains after exposure to ceftazidime in concentration MIC?2 and MIC?4, respectively. The hydrolyzing capacity of the original strains was 15-36% while the hydrolyzing capacity of the resistant strains was 10-73%. Totally 64% of the resistant strains expressed higher hydrolyzing capacity than the original strains. Conclusion. Regardless of the susceptibility test results, Ps. aeruginosa presented a high tendency to develop resistant strains after a short exposure to ceftazidime in vitro. In most cases the resistant strains expressed higher cephalosporinase activity than the original strains, suggesting derepression of chromosomal ?-lactamases. Our model offers a simple, inexpensive and rapid method for detecting resistance of Ps. aeruginosa developed due to derepression of ?-lactamases, and for discriminating resistant strains with derepressed ?-lactamases from strains that developed other mechanisms of resistance.

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