Exploring unanswered and novel questions is a cornerstone for discovery that gives immense fulfillment in life. However, the road to discovery is fraught with many seemingly insurmountable mountains. In research that includes funding, resources (intellectual and nonintellectual) and environment (appropriate infra-structure and administration that encourages and promotes).  At the fast pace at which the research technologies are changing, it has become imperative to develop an interdisciplinary and inter-institute research program. My search for intellectual and technical resources has led me to collaborate with many scientists from various disciplines locally, nationally and internationally.  The extensive list of collaborators include:

• Local: FIU - Prof Giri Narasmihan (Bioinformaticist, Computer Science), Prof John Makemson (Biochemist, Biological Sciences), Prof Brad Bennett (Ethnobotanist, Biological Sciences), Assoc Prof Krish Jayachandran (Soil Scientist, Earth Sciences), Prof Martin Quirke (Organic Chemist, Chemistry-Biochemistry), Prof Stanislaw Wnuk (Organic Chemist, Chemistry-Biochemistry), Assoc Prof Wonbong Choi (Nanomaterial Scientist, Material Science & Engineering). Non-FIU - Dr. Michael Light (University of Miami Cystic Fibrosis Center).
• National: Prof Stepehen Lory (Pseudomonas Pathogenesis Expert, Harvard Medical School), Prof & Academy Member Frederick Ausubel (Alternate Therapy & Animal Models Expert, Massachusetts General Hospital and Harvard Medical School), James Galagan  (Associate Director of Microbial Genome Analysis, Broad Institute of MIT and Harvard) and his team, Prof John Clardy (Phamacologist, Harvard Medical School), Dr. Jim Entry (Soil Scientist, USDA), Prof Kelsey Downum (Chemical Ecologist, University of Texas at Arlington), Prof Giovanni Piedimonte (Airway Inflammation Specialist, West Virginia University) and Assoc Prof Patrick Gillevet (Environmental Molecular Biologist, George Mason University)
• International: Prof Burkhard Tümmler (Cystic Fibrosis Expert, Medizinische Hochschule Hannover, Germany), Prof Soren Molin (Biofilm Expert, Danish Technical University, Denmark), Prof Niels Høiby (Cystic Fibrosis Expert, Copenhagen University, Denmark), Prof Nobuo Shimamoto (Nanobiologist, National Institute of Genetics, Mishima, Japan), Assoc Prof Balasubramanian Danajeyan (Tamil Nadu Agricultural University, India), Prof Chong-Lek Koh (Geneticist, National Institute of Education, Singapore), Prof Bola Adeniyi (Microbiologist, University of Ibadan, Nigeria), and Asst Prof Moshe Herzberg (Polymer Biologist, Ben-Gurion University of the Negev, Israel)

The major focus of my research has been to better understand microbial infections in patients with cystic fibrosis. The long-term goal is to contribute to the understanding of the pathobiology of intractable Pseudomonas aeruginosa chronic infections. The following section will discuss six areas of my research focus, (a) antibiotic resistance, (b) alginate production, (c) comparative genome analysis, (d) alternate and complementary therapy, (e) cystic fibrosis lung microbiome, and (f) soil microbiome.

Pseudomonads are environmental saprotrophs, and only Pseudomonas aeruginosa can cause serious infections in patients with cystic fibrosis (CF), chronic obstructive bronchiectasis, diffuse panbronchiolitis, pneumonia, severe burn wounds, cancer and AIDS patients. The elaboration of a myriad of virulence factors, its innate ability to counteract antibiotics and host immune response, and its resilience while undergoing numerous genetic changes to establish chronic infection have made P. aeruginosa the number one killer of CF patients. Two critical hallmarks of chronic P. aeruginosa infection are the emergence of alginate-overproducing (Alg+) mucoid colonies and the development of increased antibiotic resistance. These two areas have been the major focus of my research. In addition, we have been interested in getting a better handle on this organism by studying its genome in detail. As a consequence of their genotypic changes in the CF lungs, the P. aeruginosa strains are recalcitrant to treatment. My lab has also been interested in exploring alternative and complementary therapeutic treatments against P. aeruginosa infections. In addition, since I realized that only 5 % of microbes can be cultured, I have explored the use of various molecular tools to explore the CF lung microbiome that can potentially be used to develop molecular diagnostic tools.

Antibiotic resistance in bacteria can be contributed by many mechanisms. The one I focus on is resistance to ß-lactams (e.g., Penicillin) mediated by microbes expressing an enzyme that will hydrolyze the antibiotics, namely, ß-lactamase. The antibiotic resistant CF isolates overproduce AmpC ß-lactamase; and ampC expression requires a LysR-type global regulator, AmpR. This ampC-ampR system has been studied in other bacterial systems, but not in P. aeruginosa. This clear gap of information basically prompted me to propose to NIH to use molecular and genetic tools to better understand the role of amp genes in ß-lactam resistance and tease apart the amp transcriptional circuitry. That funding resulted in the lab reporting novel aspects of the circuitry in P. aeruginosa.

• We reported the presence of a second gene, poxB, encoding a ß-lactamase. We have shown that AmpC and PoxB contribute to the overall increase in the ß-lactamase activity and that their expression is regulated by AmpR (Kong et al., 2005b). We do not know if there is an increase in poxB transcription and if it is AmpR-mediated, though it appears AmpR-dependent. Immediately upstream of the poxAB operon is another operon that encodes a TCS (PA5511 and PA5512). We postulate that AmpR and TCS mediate regulation of the poxAB operon.
• We have reported that ß-lactam resistance involves two permeases, unlike in other systems. These two permeases were named AmpG and AmpP. AmpG appears to transport muramyl pentapeptide from the periplasm into the cytoplasm, whereas AmpP transports UDP-pentapeptide from cytoplasm to periplasm. Mutations in these two genes abolish all ß-lactamase activity. Thus, both PoxB and AmpC activities are dependent on AmpG and AmpP. AmpP-like permeases are yet to be identified in other amp systems. Though ampP appears constitutive, ampG is autoregulated. AmpR may mediate the ampG autoregulation directly or indirectly. AmpP-like permeases are yet to be identified in other amp systems.
• Though AmpR has been identified two decades ago, our lab is first to demonstrate that AmpR is a global regulator.  We demonstrated that besides antibiotic resistance, AmpR is required for normal cell growth, and it regulates pyocyanin and protease production. It is not clear if it has a direct effect or mediates by an unidentified factor. Analyses of the transcriptional activity of the genes involved show that AmpR indeed negatively regulates the transcription of the genes encoding virulent factors.

These analyses contributed to the following:

• Two NIH grants as PI since my tenure and promotion
• Role of Pseudomonas aeruginosa ß-lactamase genes (04/05 – 03/08) for $581,287.00
• Regulation of P. aeruginosa PoxB oxacillinase by AmpR and a two-component system (04/08 – 03/12) for $1,238,000  
• Three peer-reviewed publications
• Kong, K. F., Jayawardena, S. R., Del Puerto, A., Wiehlmann, L., Laabs, U., Tummler, B. & Mathee, K. (2005). Characterization of poxB, a chromosomal-encoded Pseudomonas aeruginosa oxacillinase. Gene 358, 82-92.
• K.F. Kong, S.R. Jayawardena, S. D. Indulkar, A. del Puerto, C-L. Koh, N. Høiby, and K.  Mathee.(2005) Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB b-lactamases, proteases, quorum sensing and other virulence factors. Antimicrobial Agents and Chemotherapy 49:4567-75. Article selected for Journal Highlights in the ASM News December issue. A monthly issue that highlights one article per journal that describes new and exciting developments in microbial research.
• One invited (peer-reviewed) review accepted
• K. F. Kong, L. Schneper, and K. Mathee. Beta-lactam antibiotics: from antibiosis to resistance and bacteriology. Review. Acta Pathologica, Microbiologica, et Immunologica Scandinavica
• One publications under review
• K.F Kong, A. Aguila, G. Narasimhan, J. Park, T. Ueharaa, L. Schneper, and K. Mathee. Pseudomonas aeruginosa ß-lactamase induction requires ampP and ampG encoding permeases. Molecular Microbiology.
• One PhD dissertation
• Kok Fai Kong (2005). Characterization of the amp genes involved in the regulation of β-lactamase expression in Pseudomonas aeruginosa. In Department of Biological Sciences, 283 pages. Teaching Assistantship
• Four undergraduate theses
• Alian Aguila (2003). Topology analysis of Pseudomonas aeruginosa AmpG permease. In Department of Biological Sciences, 62 pages. 
• Sergio Luna (2006). Using a novel non-radioactive method of primer extension analysis to map the transcription start sites of amp genes in Pseudomonas aeruginosa. In Department of Biological Sciences, 58 pages.
• Raquel Olavarrieta (2006) Role of AmpR in biofilm Pseudomonas aeruginosa biofilm antibiotic resistance. In Department of Biological Sciences, 40 pages.
• Raphael Bosse (2009). Role of IclR-type regulator PA4157 in ß-lactam resistance in Pseudomonas aeruginosa. In Department of Biological Sciences, 65 pages.
• Student awards (excluding numerous travel and presentation awards)
• Five Cystic Fibrosis Foundation Student Traineeships for Kok-Fai Kong (2005), Raquel Olavarrieta (2006), Diansy Zincke (2008), Deepak Balasubramanian (2009) and Raphael Bosse (2009).
• Two summer NIH-BRI-MBRS awards for Diansy Zincke (2008 and 2009).
• Alian Aguila and Diansy Zincke are recipients of MARC and RISE scholarships, respectively.
• 16 oral and poster presentation by candidate and students in local, national and international conferences and meetings.
• Current and future plans
• Currently, I have two post-doctoral fellows and one PhD student addressing the complex role of AmpR using state-of-the-art genomics and proteomics tools.
• One PhD student (Diansy Zincke, RISE Scholarship) is focusing in understanding the poxAB operon in antibiotic resistance
• Three manuscripts are nearing completion.
• I plan to resubmit a revised R01 grant.

Alginate production in P. aeruginosa is a result of mutation in a negative regulator MucA that controls a master regulator called AlgT/U. AlgT/U is a sigma factor that is necessary for transcriptional activation of all the alginate genes. In addition, over 25 genes in the pathwayhave been identified and the transcriptional regulation is very complex. However, we have made little progress in keep this process in check promoting us to take a genetic approach to find novel genes. We have identified one gene and two other cosmids that contain previously unidentified genes involved in alginate production. This work has resulted in

• Two publications under review
• K. Mathee, D. Ramos, R. Sautter, O. Ciofu, A. Heydorn, N. Hoiby, A. Kharazmi, C. DeVries, D.E. Ohman and L. Schneper. Mucoid-to-nonmucoid conversion of Pseudomonas aeruginosa occurs by multiple pathways. Gene
• R. Sautter, L. Schneper and K. Mathee. AlgO, a positive regulator of alginate production is a putative perisplamic protease with high homology to Escherichia coli Prc/Tsp. Gene
• Two MS theses
• Robert Sautter (2005). Molecular mechanism responsible for mucoid to nonmucoid conversion in Pseudomonas aeruginosa. In Department of Biological Sciences, 116 pages.
• Marios Stylianu, (2008).  Mapping mutations responsible for nonmucoid reversion phenotype of mucoid Pseudomonas aeruginosa. In Umea University, Sweden. 29 pages.
• Two undergraduate theses
• Damaris Ramos (2002). Molecular mechanism responsible for mucoid to nonmucoid conversion in Pseudomonas aeruginosa.
• Robert J. Smiddy (2005). Regulation of mucoidy in Pseudomonas aeruginosa: Identification of a novel mutation responsible for nonmucoid reversion phenotype. In Department of Biological Sciences, 42 pages.
• Student awards  (excluding numerous travel and presentation awards)
• Summer NIH-BRI-MBRS award for Robert Sautter (2005).
• Damaris Ramos, Robert Sautter and Robert Smiddy were RISE scholarship recipients
• Seven oral and poster presentations by candidate and students in local, national and international conferences and meetings.
• Current and future plans:
• Currently, I have two undergraduate students mapping two novel genes involved in alginate production.
• Plan to submit a grant to Cystic Fibrosis Foundation.

The comparative genomeanalysis was initiated during my sabbatical visit in Fall 2006 with Lory (Harvard Medical School) and Narasimhan (FIU) and many BROAD Institute fellows (http://www.broadinstitute.org/). These analyses contributed to the following:

• Funding
• As CoPI in an NIH-SCORE grant titled: SEI+II: An integrated database approach to genome-scale analysis. PI: Giri Narasimhan
• Three peer-reviewed publications
• E. Zeng, K. Mathee, and G. Narasimhan. (2007). IEM: an algorithm for iterative enhancement of motifs using comparative genomics data. Comput Syst Bioinformatics Conf.  6:227-35.
• K. Mathee, G. Narasimhan, C. Valdes, X. Qiu, J. M. Matewish , M. Koehrsen, A. Rokas, C. N. Yandava, R. Engels, E. Zeng, R. Olavarietta, M. Doud, R. Smith , P. Montgomery, J. White, P. A. Godfrey, C. Kodira, B. Birren, J. Galagan and S. Lory. Dynamics of Pseudomonas aeruginosa genome evolution. Proceedings of National Academy of Sciences USA 105(8):3100-3105. This article was highlighted by (a) Genome Technology Online, Feb 20, 2008, “Survival through genome shapeshifting” and (b) Selected for review by Faculty of 1000 Biology.
• D. Balasubramanian, and K. Mathee. Comparative transcriptome analyses of Pseudomonas aeruginosa. Human Genomics 3(4):349-361.
• 4 oral and poster presentations by candidate and students in local, national and international conferences and meetings.
• Current and future plans
• Currently, we are working on a P. aeruginosa pangenome array in collaboration with Agilent Technologies.
• We are in the process of analyzing two other new genomes, namely Burkholderia dolosa and B. cenocepia.

We approached alternate and complementary therapeutical measures in three ways. The first used an ethnobotanical approach to look at south Florida Medicinal plants for anti-infective activities. This resulted in one patent application for two compounds purified from one of the plants. This project resulted in a productive collaboration with Ausubel (Academy Member and Harvard Faculty), and Clardy (Harvard Faculty) that resulted in the purification of the compounds.
The second approach was to look at the anti-infective potential of ginseng. We sub-fractionated the active portion. Currently, we are working solving the structure of active chemicals with the help of Quirke (FIU).  Finally, in recent years in collaboration with a chemist (Wnuk, FIU), we have been working on synthetic analogs that have anti-infective activities.

• Two grants
• NIH grant (R15) as PI: Ginseng as an antibacterial agent against Pseudomonas aeruginosa. (02/05 – 01/08) for $ 207,075.00
• NIH-NCCAM grant as CoPI: Training in alternative tropical botanical medicine  (01/02 – 12/05) for $1,146.485.00
• One patent application
• Ellagitannins as inhibitors of bacterial quorum sensing. Patent application has been filed by FIU. Serial No: 61/036,812. Involved K. Mathee, A. Adonizio, F. Ausubel, J. Clardy, B. Bennett and K. Downum
• Three peer-reviewed publications
• A. L. Adonizio, K. Downum, B. C. Bennett, and K. Mathee. (2006). Anti-quorum sensing activity of medicinal plants in southern Florida. Journal of Ethnopharamocology 105:427-35.
• A. L. Adonizio, K.F. Kong, and K. Mathee. (2008).  Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrobial Agents and Chemotherapy 52:198-203.
• A. Adonizio, S. Leal, F. Ausubel, and K. Mathee. (2008). Attenuation of Pseudomonas aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model system.  Journal of Medical Microbiology 57:809-813.
• One publications under review
• KZ. Song, K.F. Kong, H. Wu, N. Høiby and K. Mathee. Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection. Phytomedicine
• One PhD dissertation
• Allison Adonizio (2008). Identification of quorum sensing inhibitors in South Florida medicinal plants. In Department of Biological Sciences, 155 pages. RISE Scholarship
• Two undergraduate theses. Both the students received Undergraduate Excellence in Research Award
• Natalie Maricic (2008). Investigating an alternative treatment to cystic fibrosis: testing the effect of Panax ginseng C.A. Meyer extracts on Pseudomonas aeruginosa. 32 pages.
• Christian Villaseca (2009). Characterization of ginseng component responsible for antiquorum sensing activity. 39 pages.
• Student awards (excluding numerous travel and presentation awards)
• Three Cystic Fibrosis Foundation Student Traineeships (two in 2005 and one 2007) and one summer NIH-BRI-MBRS award for Allison Adonizio (2005).
• Allison Adonizion is a recipient of RISE scholarship.
• 15 Oral and poster presentations by candidate and students in local, national and international conferences and meetings.
• Current and future plans
• Plan to purify the active components from ginseng and other medicinal plants.
• Demonstrate the mechanism of action of these compounds.
• Submit at least three more manuscripts.
• Apply for a R01 grant.

The CF microbiome project focused on developing tools to better understand the lung ecology in these patients that suffer from life-long chronic infection. We adopted a commonly used molecular tool to study soil microbiome to look at the complex microbial community in CF patients. This project resulted in a multidisciplinary approach involving cystic fibrosis pulmonologist (Light, UM), a computer scientist (Narasimhan, FIU) and material science engineer (Choi, FIU). The work with Choi focused on development of DNA-based sensor. These analyses contributed to the following:

• Funding
• FIU-NIH FREA Funding for $5,000 (08/06 – 12/06) as PI.
• Four peer-reviewed publications
• S. Roy S, H. Vedala, A.D. Roy, D.H. Kim, M. Doud, K. Mathee, H.K. Shin, N. Shimamoto, V. Prasad and W. Choi. (2008) Direct electrical measurements on single-molecule genomic DNA using single-walled carbon nanotubes. Nano Letters 8:26-30. Highlighted by Nanowerk Spotlight, NewScientist Tech, NewScientist, NanoScienceWorks, Technology Reviews and Nature Spotlight.
• H. Vedala, D.H. Kim, M. Doud, K. Mathee, and W. Choi (2008). Effect of environmental factors on electrical conductivity of single oligo-DNA molecule measured using single-walled carbon nanotube nanoelectrodes. Nanotechnology 19:1-6 (265704)
• M. Doud, E. Zeng, L. Schneper, G. Narasimhan, K. Mathee. Approaches to analyzing dynamic microbial communities such as those seen in cystic fibrosis lung. Human Genomics 3(3):246-256.
• M Doud, M. Light, G. Gonzalez, G. Narsimhan, and K. Mathee (2009). Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients. Human Genomics (Accepted).
• One MS thesis
• Melissa Doud (2006). The role of amplicon length heterogeneity-polymerase chain reaction in microbial community profiling and presumptive testing of bioagents. In International Forensic Research Institute, Department of Chemistry & Biochemistry, 111 pages. 
• One undergraduate thesis
• Nabil Baker (2008). Presumptive identification of fungal communities in cystic fibrosis patients sputum using amplicon length heterogeneity polymerase chain reaction. In Department of Biological Sciences, 30 pages. 
• Student awards (excluding numerous travel and presentation awards)
• One Cystic Fibrosis Foundation Student Traineeships for Melissa Doud (2008)
• One summer NIH-BRI-MBRS award for Melissa Doud (2008).
• Melissa Doud is a recipient of RISE scholarship.
• 12 oral and poster presentations by candidate and students in local, national and international conferences and meetings.
• Current and future plans
• Currently, I have one PhD student (Melissa Doud with RISE Scholarship) who is working on high-throughput state-of-the art new generation sequencing technology to sequence sputum samples for bacterial and fungal communities. This work is in part collaboration with Michael Light (UM) and Pat Gillivet (George Mason University).
• We submitted a challenge grant to study CF newborn infant microbiome in collaboration with Miami Children Hospital Cystic Fibrosis Center (Maria Franco) and FIU College Engineering (Giri Narasimhan). A revised grant will be submitted in collaboration with All Children’s Hospital in St. Petersburg, in order to increase the sample size.
• In collaboration with Adam Wanner (U of Miami) we have submitted a grant to Florida James and Esther King Biomedical Research Program that uses the same tools to look at lung microbiome of patients with chronic obstructive pulmonary disease (COPD).
• In collaboration with Chenzhong Li (FIU College of Engineering), we are in the process of developing biosensor tools that can detect multiple bacterial species in a single step.

The above microbiome work is a direct result of our work with soil microbiome using molecular metagenomic tools. This work was initiated by my ex-postdoctoral fellow DeEtta Mills (Director, DNA Fingerprinting Facility) in collaboration with Jim Entry (Soil Scientist, USDA), Krish Jayachandran (Soil Scientist, FIU Department of Earth Sciences, CAS) and Giri Narasimhan (Bioinformaticist, School of Computing, COE). We also explored the potential use of microbiome as a tool for soil identification. These analyses contributed to the following:

• Seven peer-reviewed publications
• C. Yang, E. Zeng, K. Mathee, and G. Narasimhan (2005). Querying a database of regulatory elements. Advances in Bioinformatics and its Applications: Series in Mathematical Biology and Medicine 8:81-92.
• Y. Wang, C. Yang, K. Mathee, and G. Narasimhan (2005). Clustering using Adaptive Self-Organizing Maps (ASOM) and Applications. Lecture Notes in Computer Science 3515: 944-951.
• C. Yang, D. Mills, K. Mathee, Y. Wang, K. Jayachandran, M. Sikaroodi, P. Gillevet, J. Entry, and G. Narasimhan (2006).  Ecoinformatics tools for microbial diversity studies: Supervised classification of amplicon length heterogeneity (ALH) profiles of 16S rRNA.  Journal of Microbiological Methods 65: 49-62.
• D.K. Mills, J. A. Entry, J.D. Voss, P.M. Gillevet, and K. Mathee. (2006). An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices. FEMS Microbiological Ecology 57:496-503.
• L.I.  Moreno, D. K.  Mills, J.  Entry, R.T.  Sautter, and K.  Mathee. (2006). Microbial metagenome profiling using Amplicon Length Heterogeneity-Polymerase Chain Reaction (ALH-PCR) proves more effective than elemental analysis in discriminating soil specimens. Journal of Forensic Science 51:1315-1322.
• J. Entry, D. Mills, K. Mathee, K. Jayachandran, R.E. Sojka and G. Narasimhan. (2008) Influence of irrigated agriculture on soil microbial diversity. Applied Soil Ecology 40:146-154.
• D.K. Mills, J.A. Entry, K. Mathee, and P.M. Gillevet. (2007). Mini-Review: Assessing Microbial Community Diversity Using Amplicon Length Heterogeneity PCR. Soil Science Society of America Journal 71 March-April
• Two MS theses
• Lilliana Moreno (2005). Molecular and chemical characterization of three Miami-Dade soil types for forensic comparison. In International Forensic Research Institute, Department of Chemistry & Biochemistry, 175 pages.
• Todd M. Crandall (2009). Discrimination of soils by amplicon length heterogeneity (ALH)-PCR generated bacterial community profiles; a novel application for the forensic examination of soil. In International Forensic Research Institute, Department of Chemistry & Biochemistry, 150 pages. 
• Seven oral and poster presentations by candidate and students in local, national and international conferences and meetings.
• Current and future plans
• Two manuscripts will be published based on the work by visiting scholar Balasubramanian Danajeyan (Associate Professor, Tamil Nadu Agricultural University, India) on exploitation of soil metagenomics for monitoring microbial nitrogen transformation and soil fertility.