Antimicrobial resistance is a problem facing our medical system due, in part, to evolution of resistant bacteria. In CF antibiotic resistance is more common than in the general public due to the continual administration of antibiotics given prophylactically or in response to an exacerbation. It has long been recognized that bacteria can display cross-resistance (when a bacterium develops resistance to one antibiotic it simultaneously becomes resistant to another antibiotic, even without exposure to that second antibiotic). Recent studies have demonstrated collateral sensitivity. In response to antibiotic treatment a bacterium becomes resistant, and then, in response to treatment with a different antibiotic, eventually develops resistance to that second antibiotic.

Collateral sensitivity (CS) is when a bacterium that developes resistance to a treatment antibiotic (abx #1) also becomes sensitive to a different (i.e. non-treatment antibiotic [abx #2]). In reciprocal CS, if that bacterium resistant to abx #1 is then evolved to become resistant to abx #2, it simultaneously loses resistance to abx #1. This little studied phenomenon suggests the possibility of addressing antibiotic resistance by cycling between the two antibiotics in a reciprocal CS pair. The challenge is to identify the antibiotic combinations that lead to collateral sensitivity as well as the mechanism underlying the phenomenon.

Burkholderia multivorans, a member of the Burkholderia cepacia complex (Bcc), is a Gram negative bacillus that is a pathogen often found in adults with cystic fibrosis (CF) . We are exploring collateral sensitivity and cross-resistance networks in B. multivorans and B. cenocepacia. Our goals are to 1) develop a collection of strains evolved to be resistant by serially exposing clinical isolates of Burkholderia to one of eight clinically-relevant antibiotics, 2) test resultant strains against the other seven drugs to determine collateral resistance and susceptibility patterns, 3) subject evolved strains to whole genome sequencing to identify candidate mutations and genes involved in cross-resistance and collateral sensitivity, and 4) introduce candidate mutations into wild type strains to confirm which mutations/genes are involved in CS.

To date, goals 1, 2 and 3 have been accomplished to varying degrees (see DOI:10.1089/mdr.2019.0202 and DOI: 10.1016/j.ijantimicag.2020.105994). Funding from the Cystic Fibrosis Foundation was recently obtained in collaboration with Dr. Way Sung (UNC Charlotte, Bioinformatics & Genomics Dep’t) to expand these studies to include additional antibiotics and Burkholderia species.

Change in Microbiota in CF Patients in Response to Antibiotic Treatment.

In Cystic Fibrosis (CF), a mutation in the CFTR gene results in the production of thick, sticky mucus in the lungs that is difficult to remove. Bacteria that are normally not pathogenic in the lungs grow in this mucus and cause a chronic infection and inflammatory immune response. In CF patients, there are several bacteria that are resistant to most known antibiotics, making treatment difficult. Bacteria in CF patients are usually characterized in a clinic by cultivation techniques, but culturing only identifies a fraction of bacteria.

New developments in sequencing technology are revolutionizing the study of complex microbial communities. The goals of this project are to: 1) identify indicator bacteria and viruses whose change in abundance or diversity presage onset of a pulmonary exacerbation or other changes in patient health, 2) identify bacteria and viruses that are involved in disease progression, and 3) determine how antibiotics affect the microbial population in the lung. These goals serve as the foundation for development of personalized courses of treatment to manage infections. The two main techniques used to monitor the microbiota are Illumina sequencing and qPCR.

In experiments analyzing sputum samples from a CF patient via Illumina sequencing and qPCR, the data indicate that 1) bacteria diversity can be high in CF patients not having an active infection, 2) while the presence of most taxa present in the lung change over time, a few specific pathogens are present in all samples, 3) bacterial diversity and abundance correlates with antibiotic treatment, and 4) standard antibiotic treatment does not remove all bacteria from the lung (see DOI: 10.1128/JCM.02555-14.). NIH funding was received to expand this study to 15 adolescent CF patients. Sample collection has ceased and data is currently being analyzed

Research Funding

“Identifying Collateral Sensitivity Networks and Associated Genetic Markers in Burkholderia species.” Cystic Fibrosis Foundation. $159,371, 1/15/20-7/15/21. PI: T. R Steck; Co-I: W. Sung (Bioinformatics & Genomics Dep’t, UNC Charlotte)

Recent Publications

Kavanaugh, L., Flanagan, J.N. and Steck, T.R. 2020. Reciprocal antibiotic collateral sensitivity in Burkholderia multivorans. International Journal of Antimicrobial Agents. DOI: 10.1016/j.ijantimicag.2020.105994

Flanagan, J. N., Kavanaugh, L. and Steck, T.R. 2019. Burkholderia multivorans exhibits antibiotic collateral sensitivity. Microbial Drug Resistance. DOI:10.1089/mdr.2019.0202

Flanagan, J.N. and Steck, T.R. 2018. Use of antibiotic disks to evolve drug-resistant bacteria. Antonie van Leeuwenhoek. 111(9):1719-1722. https://doi.org/10.1007/s10482-018-1055-3

Flanagan, J.N., and Steck, T.R. 2017. The relationship between agar thickness and antimicrobial susceptibility testing. Indian Journal of Microbiology. 57(4): 503-506. DOI: 10.1007/s12088-017-0683-z.

Stokell, J.R., Hamp, T., and Steck, T.R. 2016. Examining changes in bacterial abundance in complex communities using next-generation sequencing is enhanced with quantitative PCR. Antonie van Leeuwenhoek. 109(8):1161-1166. DOI: 10.1007/s10482-016-0707-4

Stokell, J.R., Gharaibeh, R.Z., Hamp, T.J. Zapata, M.J., Fodor, A.A., and Steck, T.R. 2015. Analysis of Changes in Diversity and Abundance of the Microbial Community in a Cystic Fibrosis Patient over a Multiyear Period. J. Clin. Microbiol. 53(1): 237-247. DOI: 10.1128/JCM.02555-14.

Stokell, J.R., Khan, A., and Steck, T.R. 2014. Mechanical Homogenization Increases Bacterial Homogeneity in Sputum. J. Clin. Microbiol. 52(7):2340-2345.

Stokell, J.R., Gharaibeh, R.Z., and Steck, T.R. 2013. Rapid emergence of a ceftazidime-resistant Burkholderia multivorans strain in a Cystic Fibrosis patient. J. Cystic Fibrosis 12:812-816.

Current Lab Members:

Sarah Harrison. M.S. student. Project – primary researcher on CFF funded Burkholderia project.

Tirtha Patel. Honors research student. Project – antibiotic collateral sensitivity in B. multivorans.

Enosh Ishman. Independent Investigations student. Project – identify CS systems in B. cenocepacia.

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Todd Steck