A microfluidic approach to study the effect of bacterial interactions on antimicrobial susceptibility in polymicrobial cultures

Polymicrobial infections are caused by more than one pathogen. They require antimicrobial dosing regimens that are different from those prescribed for monomicrobial infections because these interactions are predicted to influence the antimicrobial susceptibility of the individual pathogens. Here we report on a microfluidic approach to study the effect of bacterial interactions in polymicrobial cultures on the antimicrobial susceptibility. The use of microfluidics enables real-time quantification of bacterial growth dynamics in the presence and absence of antimicrobials, which is challenging to achieve using current methods. We studied microbial interactions between Pseudomonas aeruginosa, and Escherichia coli and Klebsiella pneumoniae. A key observation was that in co-cultures with relatively high initial cell numbers of P. aeruginosa, the co-cultured partner bacteria exhibited initial growth followed by lyses or growth stasis. In addition, we observed a significantly higher antimicrobial tolerance of P. aeruginosa in polymicrobial cultures, as evident by up to 8-fold increases in the minimum inhibitory concentration of the antimicrobials, compared to those observed in monomicrobial cultures. This work demonstrates the potential of microfluidics to study bacterial interactions and their effect on antimicrobial susceptibility, which in turn will aid in determining appropriate antimicrobial treatment for polymicrobial infections.

[1]  D. Oh,et al.  Libertellenones A-D: induction of cytotoxic diterpenoid biosynthesis by marine microbial competition. , 2005, Bioorganic & medicinal chemistry.

[2]  Arnab Mukherjee,et al.  A multiplexed microfluidic platform for rapid antibiotic susceptibility testing. , 2013, Biosensors & bioelectronics.

[3]  Vanessa Sperandio,et al.  Inter-kingdom signalling: communication between bacteria and their hosts , 2008, Nature Reviews Microbiology.

[4]  Albert Balows,et al.  Manual of Clinical Microbiology, 7th ed. , 2000 .

[5]  U. Reichl,et al.  Interspecies effects in a ceftazidime-treated mixed culture of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus: analysis at the single-species level. , 2011, The Journal of antimicrobial chemotherapy.

[6]  E. O'Shaughnessy,et al.  J Antimicrob Chemother 1994; 33: 350–351 , 1994 .

[7]  K. Poole.  Aminoglycoside Resistance in Pseudomonas aeruginosa , 2005, Antimicrobial Agents and Chemotherapy.

[8]  F. Lin,et al.  Recent developments in microfluidics-based chemotaxis studies. , 2013, Lab on a chip.

[9]  M. Parsek,et al.  Going local: technologies for exploring bacterial microenvironments , 2013, Nature Reviews Microbiology.

[10]  K. Rumbaugh,et al.  Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection , 2012, Proceedings of the National Academy of Sciences.

[11]  F. McKenzie.  Case mortality in polymicrobial bloodstream infections. , 2006, Journal of clinical epidemiology.

[12]  J. R.,et al.  Chemistry , 1929, Nature.

[13]  S. Eykyn.  Microbiology , 1950, The Lancet.

[14]  S. Diggle.  Microbial communication and virulence: lessons from evolutionary theory. , 2010, Microbiology.

[15]  S. Baron,et al.  Antibiotic action of pyocyanin , 1981, Antimicrobial Agents and Chemotherapy.

[16]  Vincent Gau,et al.  Antimicrobial susceptibility testing using high surface-to-volume ratio microchannels. , 2010, Analytical chemistry.

[17]  A. Griffin,et al.  Social evolution theory for microorganisms , 2006, Nature Reviews Microbiology.

[18]  M. Surette,et al.  Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication , 2003, Molecular microbiology.

[19]  William Fenical,et al.  Induced production of emericellamides A and B from the marine-derived fungus Emericella sp. in competing co-culture. , 2007, Journal of natural products.

[20]  T. Du,et al.  An explanation for the effect of inoculum size on MIC and the growth/no growth interface. , 2008, International journal of food microbiology.

[21]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[22]  Sunghoon Kwon,et al.  Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system. , 2013, Lab on a chip.

[23]  Paul J. A. Kenis,et al.  Design considerations for elastomeric normally closed microfluidic valves , 2011 .

[24]  U. Reichl,et al.  Time‐kill studies with a ceftazidime‐treated mixed culture consisting of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus , 2012 .

[25]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[26]  J. Edd,et al.  A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics , 2013 .

[27]  B. Iglewski,et al.  Bacterial Quorum Sensing in Pathogenic Relationships , 2000, Infection and Immunity.

[28]  R. Austin,et al.  Bacterial metapopulations in nanofabricated landscapes , 2006, Proceedings of the National Academy of Sciences.

[29]  F. Lépine,et al.  Active Starvation Responses Mediate Antibiotic Tolerance in Biofilms and Nutrient-Limited Bacteria , 2011, Science.

[30]  Olivier Lazcka,et al.  Pathogen detection: a perspective of traditional methods and biosensors. , 2007, Biosensors & bioelectronics.

[31]  S. Choi,et al.  Clinical significance and outcome of polymicrobial Staphylococcus aureus bacteremia. , 2012, The Journal of infection.

[32]  T. Wood,et al.  Indole Production Promotes Escherichia coli Mixed-Culture Growth with Pseudomonas aeruginosa by Inhibiting Quorum Signaling , 2011, Applied and Environmental Microbiology.

[33]  M. Burns,et al.  Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities , 2011, PloS one.

[34]  G. Bearman,et al.  Comparison of the systemic inflammatory response syndrome between monomicrobial and polymicrobial Pseudomonas aeruginosa nosocomial bloodstream infections , 2005, BMC Infectious Diseases.

[35]  L. Hoffman,et al.  Revealing the dynamics of polymicrobial infections: implications for antibiotic therapy. , 2010, Trends in microbiology.

[36]  M. Valvano,et al.  Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells , 2013, PloS one.

[37]  R. Bansal,et al.  Child Health , 1945, Nature.

[38]  D S Burgess,et al.  Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test , 1996, Antimicrobial agents and chemotherapy.

[39]  Andre Sharon,et al.  A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus aureus. , 2012, Lab on a chip.

[40]  M. Weinstein,et al.  Clinical importance of polymicrobial bacteremia. , 1986, Diagnostic microbiology and infectious disease.

[41]  R. Ismagilov,et al.  Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability. , 2009, Angewandte Chemie.

[42]  G. O’Toole,et al.  New yeast recombineering tools for bacteria. , 2009, Plasmid.

[43]  Sam P. Brown,et al.  Does multiple infection select for raised virulence? , 2002, Trends in microbiology.

[44]  G. Kaur,et al.  Clinical significance of polymicrobial bacteremia in newborns , 2005, Journal of paediatrics and child health.

[45]  F. Heilmann,et al.  Efficacy of sulbactam in an in vitro model of mixed aerobic/anaerobic infections , 1990, Infection.

[46]  Nate J. Cira,et al.  A self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics. , 2012, Lab on a chip.

[47]  J. Collins,et al.  Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance , 2013, Proceedings of the National Academy of Sciences.

[48]  H. Mobley,et al.  Complicated Catheter-Associated Urinary Tract Infections Due to Escherichia coli and Proteus mirabilis , 2008, Clinical Microbiology Reviews.

[49]  Qin Tu,et al.  High-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing in zero-flow environments. , 2011, Biosensors & bioelectronics.

[50]  Bill Bynum,et al.  Lancet , 2015, The Lancet.

[51]  Roland R. Regoes,et al.  Pharmacodynamic Functions: a Multiparameter Approach to the Design of Antibiotic Treatment Regimens , 2004, Antimicrobial Agents and Chemotherapy.

[52]  Seongyong Park,et al.  A microfluidic concentrator array for quantitative predation assays of predatory microbes. , 2011, Lab on a chip.

[53]  Yi Zheng,et al.  Recent advances in microfluidic techniques for single-cell biophysical characterization. , 2013, Lab on a chip.

[54]  Roberto Kolter,et al.  New developments in microbial interspecies signaling. , 2009, Current opinion in microbiology.

[55]  C. E. Taylor,et al.  Human polymicrobial infections , 2005, The Lancet.

[56]  S. Levy,et al.  Antibacterial resistance worldwide: causes, challenges and responses , 2004, Nature Medicine.

[57]  M. Chelius,et al.  Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association withZea mays L , 2000, Applied and Environmental Microbiology.

[58]  Arnab Mukherjee,et al.  Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters , 2013, PloS one.

[59]  A. Theberge,et al.  Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. , 2010, Angewandte Chemie.

[60]  Gerard D. Wright,et al.  Bacterial resistance to antibiotics: enzymatic degradation and modification. , 2005, Advanced drug delivery reviews.

[61]  A. Torres,et al.  Community-acquired polymicrobial pneumonia in the intensive care unit: aetiology and prognosis , 2011, Critical care.

[62]  Udo Reichl,et al.  Characterization of a three bacteria mixed culture in a chemostat: Evaluation and application of a quantitative terminal‐restriction fragment length polymorphism (T‐RFLP) analysis for absolute and species specific cell enumeration , 2007, Biotechnology and bioengineering.

[63]  L. Saravolatz,et al.  The increasing importance of polymicrobial bacteremia. , 1979, JAMA.

[64]  C. Fuqua,et al.  Bacterial competition: surviving and thriving in the microbial jungle , 2010, Nature Reviews Microbiology.

[65]  J. Montgomerie,et al.  Pseudomonas aeruginosa and Klebsiella pneumoniae on the perinea of males with spinal cord injuries , 1982, Journal of clinical microbiology.

[66]  Nate J. Cira,et al.  Rapid Identification of ESKAPE Bacterial Strains Using an Autonomous Microfluidic Device , 2012, PloS one.

[67]  D. Weibel,et al.  Rapid screening of antibiotic toxicity in an automated microdroplet system. , 2012, Lab on a chip.

[68]  R. Kolter,et al.  Interspecies chemical communication in bacterial development. , 2009, Annual review of microbiology.

[69]  M. Ferraro,et al.  Antimicrobial susceptibility testing: a review of general principles and contemporary practices. , 2009, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[70]  Int J Food Microbiol , 2011 .

[71]  J. M. Dow,et al.  Diffusible signals and interspecies communication in bacteria. , 2008, Microbiology.