Title: Associate Professor (Department of Biology
and Molecular Biology Program)
Research area: Microbiology
Office location: FH 359
Laboratory Location: FH 348/353
Email Address: firstname.lastname@example.org
Office Phone: 575-646-5660
Office Fax: 575-646-5665
Lab Webpage: http://biology-web.nmsu.edu/gustafson/GusWeb/Site/Welcome.html
- Illinois State University 1984-87. Degrees: BSc., Biology, Minor,
- Brian Wilkinson's Laboratory, Illinois State University 88-90.
Degree: M.Sc. (Microbial Physiology)
- Brigitte Berger-Bächi's Laboratory, University of
Zürich 90-94. Degree: Ph.D. (Molecular Microbiology).
- Stuart B. Levy's Laboratory, Tufts University School of Medicine
94-96. Postdoctoral fellow
My primary research plan is to continue the identification of genes that Staphylococcus aureus requires to express intrinsic and clinically-relevant resistance to various antibiotics and projects on the molecular epidemiology of MRSA in the Paso Del Norte region. My laboratories work involves the use of genome sequencing and genomic comparison, genome-wide mutational analysis, genomic microarrays, metabolomics, real-time PCR and selective capture of transcribed sequences, which are all cutting edge techniques in biology. We also utilize protein biochemistry techniques and numerous microbial physiology protocols. With regards to molecular epidemiology, students are trained in pulsed-field gel electrophoresis, locus sequence typing, and common medical microbiology techniques (plasmid profiles, drug susceptibility, toxin production, etc.) designed to scrutinize potential clonal dissemination of MRSA strains.
A Mutational “Switch” to the VISA Genotype
VISA are resistant to the action of vancomycin because of a thickened peptidoglycan layer. This thickened peptidoglycan contains elevated levels of the vancomycin target, the terminal D-ala-D-ala on the end of peptidoglycan stem peptides. The genetic mechanisms equipping these organisms with their “false-target” for vancomycin are poorly understood. We have recently discovered one hetero-VISA strain (MM66) in the ~300 MRSA strains we have thus far fingerprinted in the Paso Del Norte region. This hVISA strain is representative of the most common MRSA clone disseminating the United States and the US Paso Del Norte Region. A hVISA strain only produces high-level resistant VISA mutants when exposed to vancomycin during therapy or in vitro. Once exposed to vancomycin, our hetero-VISA produced thousands of stable VISA mutants. Interestingly, when genomic array analysis was applied to 2 separately isolated VISA mutants of our hVISA, alterations in the overall transcriptome (2,700 genes investigated) of both mutants were almost identical. We have also sent appropriate chromosomal DNA samples for genomic wide mutational analysis and now genomic sequencing and annotation. These techniques will effectively compare the entire 2.7 Mb genomes and identify all SNPs and InDels occuring in our MM66 VISA mutants, as a result of acquiring the VISA phenotype. With this work, we hope to identify the mutational “switch” to the VISA genotype that possibly can be developed into a novel antibiotic target for further investigation.
Novel steroid antimicrobial
During my first entry into research at Curtin University of Technology, I studied plasmid encoded resistance to the novel steroid antimicrobial fusidic acid. We have now identified: a fusidic acid resistance gene, far1 (historically referred to as fusB) and the fusidic acid stimulon in fusidic acid-susceptible strain SH1000. We have also characterized fusA (encoding elongation factor G) chromosomally-mediated fusidic acid-resistant mutants of Staphylococcus aureus. Two mutants demonstrated mutations in fusA as expected; the 1st-step mutant also demonstrated mutations in a putative phage protein, while the 2nd-step mutant harbored additional mutations in the accessory gene regulator gene agrA and an araC-like transcriptional regulator. Both mutants demonstrated sweeping transcriptional alterations and reduced growth rates. While some transcriptional alterations were shared between the two resistant mutants, broad profile differences were also evident in the individual mutant transcriptomes. Compared to the parent strain, both mutants demonstrated increased susceptibility to ciprofloxacin, ethidium, a pine-oil based disinfectant, alcohols and triclosan. These increased susceptibilities were attributed to: upregulation of mgrA and marR-homologues and associated downregulation of the norB and blt-like multidrug efflux pump genes; downregulation of staphyloxanthin biosynthesis genes (crtM and crtN); a gene encoding an alcohol dehydrogenase (adh1); and a gene encoding an enoyl-acyl carrier protein reductase (fabI) (-2.4 to -2.9-fold).
Household disinfectant-reduced susceptibility mechanism of S. aureus
S. aureus mutants expressing reduced susceptibility to a pine-oil based house disinfectant (POHDRS) also display reduced susceptibility to membrane denaturing antimicrobials; the cell wall-active antibiotics vancomycin and oxacillin, and the human cathepsin G peptide CG117-136. In addition, POHDRS mutants demonstrate increased anteiso fatty-acid content, altered peptidoglycan metabolism and growth rates, as wells as, reduced staphyloxanthin (orange pigment) production. Using transcriptome and comparative genomic sequencing we conclude that the POHDRS phenotype results from mutations altering the function of the catabolite control protein (ccpA) and upregulation of the mevalonate pathway and ddh, a gene previously identified to affect vancomcyin resistance levels. Furthermore, transcriptome alterations are also responsible for the altered cell wall metabolism and reduced staphyloxanthin production observed in a POHDRS mutant.
Food Matrix Growth Models
We have now produced a chicken breast growth model to examine the effects of various FDA-approved food additives on the regulation of enterotoxin production and the master virulence operon agr. Transcriptome analysis has demonstrated alterations in amino acid metabolism that allows for growth of this organism on the surface of chicken, at a rate equivalent to that observed in rich bacteriological media. This work is being extended into the development of a milk growth model. Both of these food matrixes contribute significantly to all cases of food poisoning. If we find a food additive that thwarts virulence gene production, we envision this might contribute to the food preparation industries.
- Price, C. T. D., G. W. Kaatz, and J. E. Gustafson. 2002. The multidrug efflux pump NorA is not required for salicylate-induced reduction in drug accumulation by Staphylococcus aureus. Int. J. Antimicrob. Agents 20:212-219.
- O'Brien, F. G., C. T. D. Price, W. B. Grubb and J. E. Gustafson. 2002. Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101. J. Antimicrob. Chemother. 50:313-321.
- Price C. T. D., Singh, V. K., Jayasawal, R. K., Wilkinson, B. J., and J. E. Gustafson. 2002. Pine Oil Cleaner Resistant Staphylococcus aureus: Reduced Susceptibility to Vancomycin and Oxacillin and Involvement of SigB. Appl. Environ. Microbiol. 68:5417-5421.
- Gustafson, J. E., F. G. O'Brien, M., J. Malkowski, R. F. Pfletz, W. B. Grubb and B. J. Wilkinson. 2003. Alterations in phage typing patterns in vancomycin-intermediate Staphylococcus aureus. J. Med. Micro. 52:711-714.
- O'Leary, J. O. M. J. Langevin, C. T. D. Price, J. S. Blevins, M. S. Smeltzer and J. E. Gustafson. 2004.. Effects of sarA inactivation on intrinsic multidrug resistance of Staphylococcus aureus. FEMS Microbiol. Lett. 237:297-302.
- Davis, A. O., J.O. O'Leary, A. Muthaiyan, M. J. Langevin, A. Delgado-Ramos, A.T. Abalos, A.R. Fajardo, J. Marek, B. J. Wilkinson and J. E. Gustafson. 2005. Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners. J. Appl. Microbiol. 98:364-372.
- O'Brien, F. G., T. T. Lim, D. C. Winnett, G. W. Coombs, J. C. Pearson, A. Delgado, M. J. Langevin, S. A. Cantore, L. Gonzalez, and J. E. Gustafson. 2005. Survey of El Paso Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 43:2969-2972.
- Riordan, J. T., J. O. O’Leary, and J. E. Gustafson. 2006. Contributions of sigB and sarA to distinct multiple antimicrobial resistance mechanisms of Staphylococcus aureus. Int. J. Antimicrob. Agents. 28:54-61.
- Riordan, J. T., A. Muthaiyan, W. Van Voorhies, C. T. Price, J. E. Graham, B. J. Wilkinson and J. E. Gustafson. 2007. The response of Staphylococcus aureus to salicylate challenge. J. Bacteriol. 189:220-227.
- Delgado, A., J. T. Riordan, R. Lamichhane-Khadka, D. C. Winnett, J. Jimenez, K. Robinson, f. G. O'Brien, S. A. Cantore, and J. E. Gustafson. 2007. Hetero-vancomycin-intermediate methicillin-resistant Staphylococcus aureus isolate from a medical center in Las Cruces, New Mexico. J. Clin. Microbiol. 45:1325-1329
- Lamichhane-Khadka, R., J. T. Riordan, A. Delgado, A. Muthaiyan, T. D. Reynolds, B. J Wilkinson, and J. E. Gustafson. In press 2008. Genetic changes that correlate with the Pine Oil Disinfectant-Reduced Susceptibility Mechanism of Staphylococcus aureus. J. Appl. Microbiol.
- Alejandro Delgado,, Sharear Zaman, Arunachalam Muthaiyan,
Vijayaraj Nagarajan, Mohamed O. Elasri, Brian J. Wilkinson and John E.
Gustafson. In press 2008. The Fusidic acid Stimulon of Staphylococcus aureus. J
- John E. Gustafson and Brian J. Wilkinson. 2005. Staphylococcus aureus as a food
pathogen: the staphylococcal enterotoxins and stress response systems,
(pgs 331-357) In: Understanding pathogen behaviour in food:virulence,
stress response and resistance (ed., M. Griffiths), Woodhead Publishing
Limited, Cambridge UK.
- John E. Gustafson and John D. Goldman. 2005. Epidemiology and
treatment options for select community-acquired and nosocomial
antibiotic-resistant pathogens (pgs. 387-400). In: Frontiers in
Antibiotic Resistance: A Tribute to Stuart B. Levy (eds. D. G. White,
M. N. Alekshun, and P. F. McDermott) American Society for Microbiology
Press Washington D.C.