Doctor of Philosophy
Microbiology and Immunology
Dr. Miguel A. Valvano
The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. I use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, I report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of PmB than the rest of the cells in the culture, and protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to PmB. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. I propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.
Putrescine protects from antibiotics through its ability to compete with PmB for surface binding and protection against antibiotic-induced oxidative stress. YceI proteins are conserved bacterial lipocalins or “bacteriocalins”. Bacteriocalins from different Gram-positive and Gram-negative bacteria are involved in the response to hydrophobic or amphiphilic antibiotics (PmB, rifampicin, norfloxacin and ceftazidime) but not hydrophilic ones (such as gentamicin). This effect is achieved by their preferential binding affinity to hydrophobic moieties. Together, my findings uncover a novel, non-genetic and cooperative mechanism of transient increase in resistance chemically communicated from more resistant members of heterogeneous populations to less resistant bacteria of the same or other species. This multifactorial mechanism of communication of antibiotic resistance offers novel targets for antimicrobial intervention.
El-Halfawy, Omar M., "Chemical Communication of Antibiotic Resistance by Highly Resistant Bacteria." (2014). Electronic Thesis and Dissertation Repository. 2187.