Colistin susceptibility test evaluation of multiple-resistance-level Pseudomonas aeruginosa isolates generated in a morbidostat device.

Objectives: Colistin is a last-resort antibiotic against the critical-status pathogen Pseudomonas aeruginosa. There is still uncertainty regarding how to accurately measure colistin susceptibility in P. aeruginosa. Evaluation of antimicrobial susceptibility testing (AST) methods is largely hampered by the lack of resistant isolates and those around the susceptibility breakpoint. The aim of this study was to generate such strains in a morbidostat device for use in AST method evaluation. Methods: A morbidostat device was used to cultivate susceptible clinical strains into isolates with a wide range of colistin MICs. Subsequently, five commercial AST methods were compared against the gold standard broth microdilution (BMD) method: MICRONAUT-S, SensiTest, Sensititre, Rapid Polymyxin Pseudomonas and Etest. Results: A total of 131 P. aeruginosa isolates were used for colistin susceptibility test evaluation (100 colistin susceptible and 31 colistin resistant). The 31 colistin-resistant isolates evolved resistance in the morbidostat to different MIC ranges (4-512 mg/L, 100% resistance generation efficacy). The categorical agreement (CA) rates for MICRONAUT-S, SensiTest and Rapid Polymyxin Pseudomonas were 94.7%, 93.9% and 92.4%, respectively. The Sensititre achieved the highest CA score (96.9%), whereas the Etests had the lowest CA score (84%). The very major discrepancy (VMD) rates for all tests were between 3.2% and 67.7%. Conclusions: The morbidostat device can efficiently provide laboratories with colistin-resistant strains for test evaluation. Although CA rates were high for commercial AST methods except for Etests, none met the ≤1.5% CLSI limit for VMD rates. Performance was generally inferior when using isolates with low-level resistance.

Transcriptomic Basis of Serum Resistance and Virulence Related Traits in XDR P. aeruginosa Evolved Under Antibiotic Pressure in a Morbidostat Device.

Colistin is a last resort antibiotic against the critical status pathogen Pseudomonas aeruginosa. Virulence and related traits such as biofilm formation and serum resistance after exposure to sub-inhibitory levels of colistin have been underexplored. We cultivated P. aeruginosa in a semi-automated morbidostat device with colistin, metronidazole and a combination of the two antibiotics for 21 days, and completed RNA-Seq to uncover the transcriptional changes over time. Strains became resistant to colistin within this time period. Colistin-resistant strains show significantly increased biofilm formation: the cell density in biofilm increases under exposure to colistin, while the addition of metronidazole can remove this effect. After 7 days of colistin exposure, strains develop an ability to grow in serum, suggesting that colistin drives bacterial modifications conferring a protective effect from serum complement factors. Of note, strains exposed to colistin showed a decrease in virulence, when measured using the Galleria mellonella infection model. These phenotypic changes were characterized by a series of differential gene expression changes, particularly those related to LPS modifications, spermidine synthesis (via speH and speE) and the major stress response regulator rpoS. Our results suggest a clinically important bacterial evolution under sub-lethal antibiotic concentration leading to potential for significant changes in the clinical course of infection.