(131d) Microfluidic Approach for Antibiotic Susceptibility Testing of Polymicrobial Cultures

Presence of multiple bacteria in several clinically important infections (polymicrobial infections), such as urinary tract infections, peritonitis, abdominal sepsis, and aspiration pneumonia, are known to have higher mortality rate than monomicrobial infections such as tetanus or typhoid fever [1, 2].  Polymicrobial antibiotic susceptibility testing (AST) is not well studied, even though bacterial interactions are known to affect the AST outcome [3].  Hence, inappropriate antibiotic treatments in polymicrobial infections are often a major cause of mortality.  Prior studies have relied on tedious methods such as quantitative terminal restriction fragment length polymorphism (qt-RFLP) and conventional plating methods that are time consuming and qualitative [4].  Following our prior work on AST using E. coli as model pathogen [5], we now extend our microfluidic approach to quantify bacterial interactions in polymicrobial infections and determine susceptibility of bacteria in polymicrobial communities against commonly used antibiotics. 

 Specifically, we utilized a 4x12-array multiplexed microfluidic platform for studying and quantifying antibiotic susceptibility of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Burkholderia multivorans in mixed cultures.  Green and red fluorescent reporters were used to distinguish bacteria in a mixed culture and time lapse fluorescence microscopy was used for direct visualization and quantification of cell numbers over long periods of time.  In a mixed culture of P. aeruginosa and E. coli, we observed lysis of E. coli and decreased growth rates of P. aeruginosa by approximately 20-40 minutes.  In addition, we compare the minimum inhibitory concentration (MIC) of antibiotics against bacteria in mixed cultures with their constituent bacterial isolates.  Interestingly, the MIC of antibiotics against P. aeruginosa increased drastically in presence of E. coli and K. pneumoniae.  For example, the MIC of P. aeruginosa against tobramycin increased by a factor of 8 mixed with E. coli.  In our ongoing work, we are performing similar experiments with bacteria including A. baumannii and B. multivorans.

This microfluidic approach offers several advantages such as rapid turn-around times (2-4 hours vs. 2-4 days), reduced sample volumes (~ 2 nL vs. 30 mL), and high detection sensitivity (1 cell vs. 10⁶ cells) over current assay methods.  Most importantly, our results prove that significant differences exist in susceptibility of bacteria in polymicrobial cultures vs. monomicrobial cultures that can be rapidly diagnosed and studied using our unique diagnostic platform for a largely understudied but clinically important field of polymicrobial infections.

[1] F. Heilmann, Infection, 1993, 3, 187-190.
[2] A.R. Marra et al., BMC Infectious Diseases, 2005, 5, 1471-1565
[3] C. Riedele and U. Reichl, Eng Life Sci., 2012, 12,188-97
[4] J.K. Schmidt et al., Biotechnol Bioeng, 2007, 96, 738-56
[5] R Mohan et al., Biosensors and Bioelectronics, 2013 (Accepted)