J. M. Whichard, K. Joyce,
P. D. Fey, J. M. Nelson, F. J. Angulo, and T. J. Barrett.
2005. Extended-spectrum
ß-lactam
Resistance among Human Clinical
Enterobacteriaceae in the United States: Results and Characterization of
2000 NARMS Surveillance. Emer. Infect. Dis. 11:1464-1466.
S.
Kozitskaya, M. E. Olson, P. D. Fey, W. Witte, K. Ohlsen, and W.
Ziebuhr. 2005. Clonal
Analysis of Biofilm-positive and Biofilm-negative Staphylococcus epidermidis
Isolates by Multilocus Sequence Typing (MLST). J. Clin. Microbiol.
43:4751-4757.
Rupp ME, Fitzgerald T, Marion N, Helget V, Puumala S,
Anderson JR, and Fey P.D. 2004. Effect of silver-coated urinary
catheters: efficacy, cost-effectiveness, and antimicrobial resistance.
Am J Infect
Control. 32:445-450
Z. Yang, J. Kovar, J. Kim, J. Niefteldt, D.R. Smith, R.
Moxley, P.D. Fey, and A.K. Benson. 2004. Identification of frequent
subpopulations of sorbitol-non-fermenting, beta-glucuronidase-negative
Escherichia coli O157:H7 from bovine production environments and human
clinical samples. Applied and Environmental Microbiology, 70:6846-6854.
W.P. Giles,
A.K. Benson, M.E. Olson, J.M. Whitchard, R.W. Hutkins, P.L. Winokur, and
P.D. Fey. 2004. DNA sequence analysis of regions surrounding blacmy-2
from multiple Salmonella plasmid backbones. Antimicrob. Agents
and Chemother. 48:2845-2852.
M.M. Samrakandi, C. Zhang, M. Zhang, J. Nietfeldt, G. Duhamel,
M.E. Olson, P.D. Fey, P.C. Iwen, S.H. Hinrichs, J.D. Cirillo, and A.K.
Benson. 2004. Genome diversity among Francisella tularensis
subspecies tularensis and Francisella tularensis subspecies
holarctica in North America. FEMS Microbiol Lett. 237:9-17.
L.D. Handke, K.M. Conlon, S.R. Slater, S. Elbaruni, F.
Fitzpatrick, H. Humphreys W.P. Giles, M.E. Rupp, P.D. Fey and J.P.
O’Gara. 2004. Genetic and Phenotypic analysis of phenotypic variation in
multiple Staphylococcus epidermidis isolates. Journal of Medical
Microbiology 53:367-374.
Fey, P.D.
and M.E. Rupp. 2003. Molecular epidemiology in the public health and
hospital environments. Clin Lab Med 23:885-901.
Tarkin IS, Henry TJ, Fey PD, Iwen PC, Hinrichs SH, and
Garvin KL. 2003. PCR rapidly detects methicillin-resistant staphylococci
periprosthetic infection. Clin Orthop 414:89-94.
Rupp, M.E., S.J. Medcalf, P.D. Fey, L.D. Handke, and
N.D. Marion. 2003. Monsel’s solution: a potential vector for nosocomial
infection. Infect. Control Hosp. Epidemiol. 24:142-144.
Rupp, M.E.
and P.D. Fey. 2003. Extended-spectrum
-lactamase
(ESBL)-producing Enterobacteriaceae. Considerations for Diagnosis,
Prevention and Drug Treatment. Drugs. 63: 353-365.
Fey, P.D.,
S. Saïd-Salim, M.E. Rupp, S.H. Hinrichs, D. J. Boxrud, C.C. Davis, B.N.
Kreiswirth, and P.M. Schlievert. 2003. Comparative molecular analysis of
community-acquired methicillin-resistant Staphylococcus aureus and
hospital-acquired methicillin-resistant Staphylococcus aureus.
Antimicrob. Agents Chemother. 47:196-203
Carattoli, A., Tosini, F., W.P. Giles, Rupp, M.E., Hinrichs,
S.H., Angulo, F.J., Barrett, T.J., and Fey, P.D. 2002.
Characterization of plasmids carrying CMY-2 from expanded-spectrum
cephalosporin-resistant Salmonella isolated in the United States
between 1996 and 1998. Antimicrob. Agents Chemother. 46:1269-1272.
Rupp, M.E., Fey, P.D.
In vivo
models to evaluate adhesion and biofilm formation by Staphylococcus
epidermidis. In Methods in Enzymology. 2001, vol. 336; pp. 206-215.
Academic Press, New York, NY
Fowler, V.G., P.D. Fey, L.B. Reller, A.L. Chamis, G.R.
Corey, and M.E. Rupp. 2001. The intercellular adhesin locus ica is
present in clinical isolates of Staphylococcus aureus from bacteremic
patients with infected and unifected prosthetic joints. Med. Microbiol.
Immunol. 189:127-131.
Rupp, M.E., N. Marion, P.D. Fey, D.L. Bolam, P.C. Iwen,
C.M. Overfelt, L. Chapman. 2001. Outbreak of vancomycin-resistant
enterococci in a neonatal intensive care unit. Infect. Control Hosp. Epi.
22:301-303.
Rupp, M.E., P.D. Fey, and G.M. Longo. 2001. Effect
of LY333328 against vancomycin-resistant Enterococcus faecium in a
rat central venous catheter-associated infection model. J. Antimicrob.
Chemother. 47:705-707.
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The Fey laboratory is
part of the staphylococcal research group within the Department of Pathology
and Microbiology (including Drs. Bayles, Dunman, Fey, Hinrichs, Rupp, and
Somerville)
Staphylococcus epidermidis
is the preeminent cause of
biomaterial-related infections and is a significant cause of morbidity and
mortality in hospital settings. In contrast to S. aureus, which
produces an array of toxins and adherence factors, the most significant
virulence factor associated with S. epidermidis
biomaterial-related infections is the synthesis of large amounts of biofilm,
which is largely composed of polysaccharide intercellular adhesin (PIA).
PIA, which is synthesized by enzymes encoded by the four gene icaADBC
operon, dramatically reduces the effectiveness of antibiotics and the host
immune system.
As a
result of current studies we propose that there are at least three stages of
biofilm development that are crucial to S. epidermidis pathogenesis.
First, initial attachment to the surface is mediated through specific
adhesins such as AtlE and fibrinogen binding protein. Indeed, work from our
laboratory demonstrated that atlE mutants are less virulent than
wild-type in a rat-catheter model of infection. The second step in
biofilm development involves intercellular aggregation and cellular
proliferation mediated by PIA. PIA production is regulated by multiple
factors including IcaR, SarA, and the alternative transcription factor
sB.
We have shown the significance of PIA
in biomaterial related infections in both rat and mouse animal models where
icaADBC mutants are less virulent in vivo. The third step, which we
believe is underappreciated, is the maintenance of a biofilm. Beenken et al
have demonstrated that at least 132 genes were differentially regulated in a
S. aureus biofilm as compared to both exponential and stationary
phase growth suggesting that other biological processes also play an
important role in either the establishment, maintenance or stability of a
mature biofilm. Similarly, Otto and colleagues postulate that phenol
soluble modulins (PSMs) are necessary for loosening the upper layers of a
S. epidermidis biofilm; mutants that lack PSM production have altered,
thicker biofilm. In addition, data presented in this application suggests
that the arginine deiminase system (ADS) is important in the development and
maturation of a biofilm. Collectively, these data suggest that all three
aspects of biofilm development (adherence, production of PIA, maintenance)
are needed to ensure maturation of a biofilm.
S.
epidermidis undergoes a phenomenon termed
phenotypic variation whereby a certain proportion of the population does not
produce PIA. Data from our laboratory suggests that phenotypic variation
due to mutations within loci responsible for PIA production (33) (data
presented in this application). In-vitro flow cell biofilm experiments have
linked the appearance of phenotypic variants to the development and
maturation of a biofilm including the appearance of “tower structures” and a
shift in metabolic activity (see preliminary studies section). In addition,
S. epidermidis GeneChip analysis demonstrates that transcription of a
umuC/uvrX/yqjW-like gene (DNA polymerase V-hereafter referred
to as umuC), an error-prone polymerase that is part of the recA-mediated
SOS regulon, is upregulated during the maturation of a biofilm (see
preliminary studies section). Both recA and umuDC have been
linked to stress-induced mutagenesis. We hypothesize that phenotypic
variation is a by-product of biofilm maturation, is biologically significant
with respect to maintenance of a biofilm, and is regulated by recA
activation. Another hallmark of biofilm maturation observed in the flow
cell experiments is the metabolism of arginine. When glucose is limited,
arginine is catabolized in the staphylococci by the arginine deiminase
system (ADS). The ADS catalyzes the conversion of arginine to ornithine,
ammonia and carbon dioxide while generating 1 mol of ATP per mol of arginine.
Preliminary data presented in this application demonstrates the following:
A) There is a direct correlation between arginine utilization, biofilm
maturation, and phenotypic variation B) ADS genes are significantly
upregulated as a S. epidermidis biofilm matures. C) An association
exists between isolates obtained from disease and two complete gene
clusters of the ADS as compared to commensal S. epidermidis isolates
obtained from healthy donors (which typically carry one gene cluster of the
ADS). We propose that arginine catabolism is
crucial to S. epidermidis biofilm maturation, development, and
therefore, the ability of S. epidermidis to establish a
biomaterial-related infection.
Finally, mutants that are defective in icaADBC transcription (icaADBC,
sarA, or sigB) produce unique biofilms in an in-vitro flow cell
and are phenotypically distinct from wild-type (icaADBC positive)
biofilms. Previous data has shown that icaADBC transcription is
negatively regulated by IcaR and positively regulated by SarA. In addition,
the alternative sigma factor sB
regulates
icaADBC transcription through repressing icaR transcription. The
molecular link between
sB
and icaR
transcription is not known. In addition, the presence of ethanol can
activateicaADBC transcription through repression of icaR
transcription in a
sB-independent
manner. We propose to use transposon mutagenesis
and GeneChip analysis to characterize the molecular link between
sB
and
ethanol repression of icaR transcript. The central hypothesis of our
work is that PIA production and biofilm maintenance /maturation are vital to
S. epidermidis pathogenesis.
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