Isolation and Characterization of Small Colony Variants of Pseudomonas aeruginosa from Monomicrobial and P. aeruginosa-Aspergillus fumigatus Mixed Microbial Biofilms

Ref ID: 18748

Author:

E. K. Manavathu, Ph. D. – Research Scientist, D. L. Vager, M. S. – Med. Lab. Technologist, J. A. Vazquez, M. D. – Senior Staff;

Author address:

Henry Ford Hosp., Detroit, MI.

Full conference title:

52nd Annual ICAAC

Date: 9 September 2014

Abstract:

Background: Small colony variants (SCVs) of P. aeruginosa (Pa) are isolated from the airways of cystic fibrosis (CF) patients. The SCVs are characterized by their slow growth, resistance to antimicrobials, wrinkled colony morphology, enhanced biofilm production and the ability to establish chronic infection. The emergence of SCVs is believed to be a stress response for survival in a hostile environment. Since the coexistence of Pa and A. fumigatus (Af) in polymicrobial (PM) biofilm provides a hostile ecological niche for these natural enemies, we investigated the prevalence and characteristics of Pa SCVs in monomicrobial (MM) and PM biofilms. Methods: MM and PM biofilms of Pa and Af were grown in 6-well tissue culture plates for 48 h in SD broth at 35 ºC. The biofilms were washed, resuspended and aliquots were plated on BHI agar containing 1 µg/ml ciprofloxacin (CIP). The plates were incubated at 35 ºC for 24 h, followed by 3-4 days at room temperature for the development of SCVs. Colony morphology was assessed by microscopy and the drug susceptibilities of planktonic cells, MM and SCV-Af biofilms were determined as previously described. Results: SCVs from the MM and PM biofilms were isolated at a frequency of 1.5 x 10-8 and 1 x 10-7 (P<0.05), respectively. Microscopic examination of the SCVs showed that 20% had wrinkled colony, whereas 80% had smooth colony morphology. The MICs of cefepime, CIP and tobramycin (TOB) for the SCVs were 8-16 fold higher (MIC 4-8 µg/ml) than that of the parent strain (MIC 0.5-1 µg/ml). A comparison of the susceptibilities of SCV MM (2-3 log CFU reduction at 64 µg/ml) and SCV-Af PM (2.5- 3 log CFU reduction at 64 µg/ml) biofilms to CIP and TOB with those of the parent strain (5-7 log CFU reduction at 64 µg/ml) showed that the SCV biofilm is less susceptible (P<0.001) to antimicrobials. Conclusions: The persistence of Pa in chronic infections in the airways of CF patients may be multifactorial, but at least partly due to SCVs that are characteristically different from non-small colony producers.

Abstract Number: M-986

Conference Poster: y

Conference Year: 2012

Link to conference website: NULL

New link: NULL

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