Left to right: Joanne Dupre (Ph.D. Scholar); Tracey Reynolds (Howard
Hughes Undergraduate Research Scholar); Reena Lamichhane-Khadka (Ph.D.
Scholar); John E. Gustafson; and Alejandro Delgado (NIH-Research Initiative
for Scientific Enhancement Scholar, Ph.D. Scholar).
Gustafson Laboratory: Microbe Model
Staphylococcus aureus is the leading cause of nosocomial
infections, bacteremias, and surgical wound infections. In 2006, methicillin-resistant S. aureus (MRSA)
were the leading cause of integumental infections in patients presenting to
emergency rooms in the United States. S. aureus can also produce numerous enterotoxins that act on the
emetic response, and is responsible for upwards of 185,000 cases of food
poisoning each year. S. aureus
endocarditis is 100% fatal in the absence of antibiotic therapy and during
the preantibiotic era, bacteremia caused by this organism reached fatality
rates of 80%. Even with effective antibiotic therapies, mortality due to S.
aureus bacteremia can still be as high
as 32%. The New York metropolitan area reported 13,550 S. aureus infections in 1995 that led to 1,400 mortalities and
costed $435.5 million dollars. Approximately 50% of MRSA identified in US
hospitals are susceptible only to the glycopeptide antibiotic vancomycin. Multiple
antibiotic-resistant MRSA (resistant to ≥ 3
antibiotic classes) are commonly isolated from patients, and
vancomycin-intermediate (1997) and -resistant (2002) S. aureus (VISA and VRSA respectively) have recently emerged. VISA
strains are now becoming common and many clinical researchers believe that
like the advent of MRSA, we are just seeing “the tip of the iceberg”.
Overall, S. aureus represents one of
the demonically perfect Gram-positive human pathogens. Introduction to Research 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 arrays,
metabolomics, real-time PCR and selective capture of transcribed sequences,
which are all cutting edge techniques in molecular 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. Research Projects
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. This work is being performed in collaboration with my former
Illinois State University Undergraduate and Masters Research Mentor, Professor Brian J. Wilkinson. Novel steroid antimicrobial During my first entry into research at Curtin University of
Technology, I was encouraged by Professor Warren Grubb to study 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 acessory 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 FusR
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 S. aureus is one of the
leading causes of mastitis in cattle and also leads to millions of dollars in
lost revenue each year for the milk industry. 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.
Recent laboratory publications: 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. Presence of
a 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. Antimicrob. Chemother. 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. Working in a BSL-2 Laboratory All
students considering the pursuit of research in my laboratory must first read
the regulations required to work in a Biosafety Level-2
laboratory The Ideal Graduate Student I
am only looking for potential Graduate students who understand my
research interests, have read my publications, and have their own ideas as to
where my research efforts should be directed. Individuals applying to my
laboratory do not have to be experts at the techniques that we utilize day to
day, but they must understand the theory behind them and their application.
These individuals must also work well with a diverse group of young
scientists and work extremely hard to finish their NMSU research education. Graduate
students are required to be able to complete and submit an internationally
recognized Journal article before graduation from my laboratory. A complete
Ph.D. thesis should be able to generate at least 3 primary publications and a
Masters thesis should generate at least 1. Graduate students can apply to work in my laboratory in
the Biology Department, or through the NMSU Graduate Program in Molecular
Biology. NMSU Graduate
Program in Molecular Biology I encourage you to contact me while applying. The Ideal Undergraduate Student I
encourage all NMSU undergraduates to get "hands on" training in the
sciences. I am looking for individuals who are willing to begin under the
guidance of Graduate students. This association should eventually allow the
undergraduate researcher to find a niche for themselves for independent
research efforts. An undergraduate project tends to be part of a larger
whole, but I make every effort to include credit for their work in
presentations and publications. How far you go in my laboratory depends
solely on your individual work ethic and leadership. Graduate students at New Mexico State University: 1. Jessica O. O'Leary (MSc.) 02-04.The role of sarA and sigB in multidrug resistance and house
cleaner tolerance in S. aureus. (Biology Educator NMSU). 2. David Winnett (MSc.) 03-05. Further
characterization of house-cleaner reduced susceptibility mutants of Staphylococcus
aureus. (Private environmental
agency). 3. Stephanie Chapman (MSc.) 03-06 Non-thesis option. (Sandia laboratories, Technician) 4. Skye Riorden (Ph.D) 03-06. Characterization of
chromosomally-encoded intrinsic multidrug resistance in S. aureus. Awarded NMSU Graduate
Assistantship award 2005-06. (Postdoctoral Scholar. Michigan State
University). 5. Alejandro Delgado (MSc. RISE student) 04-06.
Epidemiology of S. aureus causing infections in two Las Cruces area hospitals.
(Ph.D. RISE student) 2004-06. Analysis of chromosomally- mediated fusidic
acid resistance mechanism(s) of Staphylococcus aureus. 6. Stephanie Cantore (MSc.) 04-06. The effects of sarA on the vancomycin intermediate
resistance mecchanism of S. aureus, and the effects of the non-steroidal antiiflammatory
diclofenac on S. aureus antibiotic resistance. (El Paso High School Teacher) 7. Reena Lamichhane-Khadka (Ph.D.) 05 -. Genes
required for chromosomally-encoded
intrinsic multidrug resistance in S. aureus. 8. Joanne Dupre (Ph.D.) 05 -. Characterization of strains
of Staphylococcus aureus isolated from Paso Del Norte dairy milk cows. 9. Jesus Cuaron (Ph.D.) (RISE) 07
-. 10. Jasmine Pando (Masters) (AMP)
07- Undergraduate research students at New Mexico State
University: 1. Austin Davis 02-03; 2. Eduardo Arozarena - 02; 3. Dave Winnett
02; 4. Alisha Fajardo 02-03
(Optometry School); 5. Brigitte Love 02-03 (RISE, Nurse); 6. Stephanie
Chapman 02-03 (MARC student); 7. Andrew Abalos 02-04 (MARC) (University of
Arizona, Epidemiology Ph.D. Program), 8. Mark Langevin 02-03 (Western Canadian College of Veterinary Medicine,
University Of Saskatchewan), 9. Alejandro Delgado 02-03 (RISE
student), 10. Jacqueline Marek 03 (RISE/MARC student); 11. Matt Shepardson
2003; 12. Kara Taylor 04 (UNM Pharmacy School), 13. Sherry Kamali 04 (NMSU
Student Reagents 03-06); 14. Anna Huerta 04 (RISE) (University of Texas El
Paso Physical Therapy School), 15. Cristina Conklin 04 (University of New
Mexico School of Medicine); 16. Stephanie Cantore (Centers for Disease
Control Internship) 04; 17. Tovarai Tso 04 (BRIDGES); 18. Kathrynn Girard,
04; 19. Sarah Dueñas (MARC student) 05-06; 20. Alesha
Elizabeth Ann Norman (MARC) 06; 21. Zena Archie 06 (BRIDGES); 22.
Sonia Horan 06 (Washington State University); 23. Shahrear Zaman (HHMI
Scholar) 06-; 24. Tracey Reynolds (HHMI Scholar) 06-; 25. Adele Garrison
(HHMI Scholar) 07 - 26. Danielle Goldtooth (BRIDGES) 07; 27. John Rivera
(MARC student); 28. Admed Manshad Graduate and Honours students at Curtin University
of Technology: 1. Adell N. Clair (Honors) 97. Characterisaton of
salicylate resistant Escherichia coli. (Physical Therapist). 2. Frances O’Brien (Ph.D.) 97-04. Cloning of fusidic acid
resistance genes from Staphylococcus aureus. (Head Investigator at
Gram-positive typing center Curtin University of Technology/Royal Perth
Hospital). 3. Christopher Price (Honours) 98. Effects of salicylate
and related compounds on fusidic acid resistance and cell membrane parameters
in Staphylococcus aureus. (Ph.D.) 99-03. Intrinsic multidrug resistance in Staphylococcus
aureus.
(Postdoctoral Fellow University of Louisville School of Medicine). 4. Prerna Rajput (Honours) 98. Effects of salicylate and
related compounds on vancomycin resistance, cell growth and autolysin production
and activity in Staphylococcus aureus. 5. Pierre Candelaria (Honours) 99. Conjugation of
vancomycin resistance determinant vanA from Western Australian poultry Enterococcus
faecalis isolates
into human E. faecalis isolates. (Ph.D., Institute of Child Health
Research, Perth, Western Australia) Undergraduate research students at Curtin University
of Technology: 1. Stephanie Gunn 97; 2. Christopher Price 97; 3. Prerna
Rajput 96-97; 4. Bradley Patrick Shelton 1996-97 (Ph.D.
Curtin University of Technology); 5. Tracy
McWilliams 97-98 (Ph.D. Investigator at Gram-positive typing center
Curtin University of Technology/Royal Perth Hospital);
6. Pierre Candelaria 97-98; 7. Scott Fisher 1997-98 (Postdoctoral Fellow); 8.
Jodie Goodridge 97-99; 9. Soo Erh Chew 99; 10. Briohny Callaghan 99;
11. Craig McClure 99; 12.
Natasha Rodrigues 99; 13. Daniel Robson 99; 14. Aaron D’souza 99; 15. Marnie
Sindu 99; 16. Raylene Louisa Lim 96 (Postdoctoral Fellow); 17. Belinda
Meiling Kong 96; 18. Tanya Lichocik 96; 19. Emma Irene Waddington 96; 20.
Nyree Dale Phillips 96; 21. Cameron Ian Botterill 96; 22. Travis Graeme
Endersby 96; 23. Niki Foster 98; 24. Sharon Szetafic 98; 25. Michael Banazis
00 (Murdoch University, Western Australia, Ph.D. Program). GRAM-POSITIVE
LABORATORY MISSION STATEMENT WE
INTEND TO COMPLETE INTERNATIONALLY RECOGNIZED RESEARCH PROJECTS ON
GRAM-POSITIVE PATHOGENS AND PUBLISH AS MANY MANUSCRIPTS IN INTERNATIONALLY
RECOGNIZED JOURNALS AS POSSIBLE. FURTHERMORE, WITH A STRONG RESEARCH FOUNDATION,
WE WILL BECOME SUCCESSFUL AT SECURING COMPETITIVE GRANTS TO FUND OUR RESEARCH
ACTIVITIES AND TRAIN DESERVING NMSU STUDENTS. |