[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Multiple-Drug Resistant Enterococci:  The Nature of the Problem and an
Agenda for the Future

Mark M. Huycke,* Daniel F. Sahm, and Michael S. Gilmore
*University of Oklahoma Health Sciences Center, Oklahoma, USA; Department
of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA; and MLR
Pharmaceutical Services, Inc., Reston, Virginia, USA

  --------------------------------------------------
      Enterococci, leading causes of nosocomial bacteremia, surgical
      wound infection, and urinary tract infection, are becoming
      resistant to many and sometimes all standard therapies. New
      rapid surveillance methods are highlighting the importance of
      examining enterococcal isolates at the species level. Most
      enterococcal infections are caused by Enterococcus faecalis,
      which are more likely to express traits related to overt
      virulence butfor the momentalso more likely to retain
      sensitivity to at least one effective antibiotic. The remaining
      infections are mostly caused by E. faecium, a species virtually
      devoid of known overt pathogenic traits but more likely to be
      resistant to even antibiotics of last resort. Effective control
      of multiple-drug resistant enterococci will require 1) better
      understanding of the interaction between enterococci, the
      hospital environment, and humans, 2) prudent antibiotic use, 3)
      better contact isolation in hospitals and other patient care
      environments, and 4) improved surveillance. Equally important
      is renewed vigor in the search for additional drugs,
      accompanied by the evolution of new therapeutic paradigms less
      vulnerable to the cycle of drug introduction and drug
      resistance.

The past few years have witnessed increasing interest in enterococci. Until
recently, these ordinary bowel commensals languished as misclassified
streptococci, commonly perceived "with the exception of endocarditis and
rare cases of meningitis  [as] not  a major cause of serious infection"
(1). In the last decade, however, enterococci have become recognized as
leading causes of nosocomial bacteremia, surgical wound infection, and
urinary tract infection (2,3). Two types of enterococci cause infections:
1) those originating from patients' native flora, which are unlikely to
possess resistance beyond that intrinsic to the genus and are unlikely to
be spread from bed to bed, and 2) isolates that possess multiple antibiotic
resistance traits and are capable of nosocomial transmission. The
therapeutic challenge of multiple-drug resistant (MDR) enterococcithose
strains with significant resistance to two or more antibiotics, often
including, but not limited to, vancomycinhas brought their role as
important nosocomial pathogens into sharper focus.

The accretion and spread of antibiotic resistance determinants among
enterococci, to the point where some clinical isolates are resistant to all
standard therapies, highlight both the vulnerability of our present
armament as well as the looming prospect of a "postantibiotic era" (4).
This review focuses on the magnitude and nature of the problem posed by
enterococci in general, and MDR enterococci in particular. For many points,
only representative citations are provided.

Habitat and Microbiology

Enterococci normally inhabit the bowel. They are found in the intestine of
nearly all animals, from cockroaches to humans. Enterococci are readily
recovered outdoors from vegetation and surface water, probably because of
contamination by animal excrement or untreated sewage (5). In humans,
typical concentrations of enterococci in stool are up to 10(sup 8) CFU per gram
(6). Although the oral cavity and vaginal tract can become colonized,
enterococci are recovered from these sites in fewer than 20% of cases. The
predominant species inhabiting the intestine varies. In Europe, the United
States, and the Far East, Enterococcus faecalis predominates in some
instances and E. faecium in others (6). Ecologic or microbial factors
promoting intestinal colonization are obscure. Of 14 or more enterococcal
species (7), only E. faecalis and E. faecium commonly colonize and infect
humans in detectable numbers. E. faecalis is isolated from approximately
80% of human infections, and E. faecium from most of the rest. Infections
to other enterococcal species are rare.

Enterococci are exceedingly hardy. They tolerate a wide variety of growth
conditions, including temperatures of 10C to 45C, and hypotonic,
hypertonic, acidic, or alkaline environments. Sodium azide and concentrated
bile salts, which inhibit or kill most microorganisms, are tolerated by
enterococci and used as selective agents in agar-based media. As
facultative organisms, enterococci grow under reduced or oxygenated
conditions. Enterococci are usually considered strict fermenters because
they lack a Kreb's cycle and respiratory chain (8). E. faecalis is an
exception since exogenous hemin can be used to produce d, b, and o type
cytochromes (9,10). In a survey of 134 enterococci and related
streptococci, only E. faecalis and Lactococcus lactis expressed
cytochrome-like respiration (11). Cytochromes provide a growth advantage to
E. faecalis during aerobic growth (9). E. faecalis cytochromes are only
expressed under aerobic conditions in the presence of exogenous hemin
(9,10,12) and, therefore, may promote the colonization of inappropriate
sites.

Enterococci are intrinsically resistant to many antibiotics. Unlike
acquired resistance and virulence traits, which are usually transposon or
plasmid encoded, intrinsic resistance is based in chromosomal genes, which
typically are nontransferrable. Penicillin, ampicillin, piperacillin,
imipenem, and vancomycin are among the few antibiotics that show consistent
inhibitory, but not bactericidal, activity against E. faecalis. E. faecium
are less susceptible to -lactam antibiotics than E. faecalis because the
penicillin-binding proteins of the former have markedly lower affinities
for the antibiotics (13). The first reports of strains highly resistant to
penicillin began to appear in the 1980s (14,15).

Enterococci often acquire antibiotic resistance through exchange of
resistance-encoding genes carried on conjugative transposons,
pheromone-responsive plasmids, and other broad-host-range plasmids (6). The
past two decades have witnessed the rapid emergence of MDR enterococci.
High-level gentamicin resistance occurred in 1979 (16) and was quickly
followed by numerous reports of nosocomial infection in the 1980s (17).
Simultaneously, sporadic outbreaks of nosocomial E. faecalis and E. faecium
infection appeared with penicillin resistance due to -lactamase production
(18); however, such isolates remain rare. Finally, MDR enterococci that had
lost susceptibility to vancomycin were reported in Europe (19,20) and the
United States (21).

Among several phenotypes for vancomycin-resistant enterococci, VanA
(resistance to vancomycin and teicoplanin) and VanB (resistance to
vancomycin alone) are most common (22). In the United States, VanA and VanB
account for approximately 60% and 40% of vancomycin-resistant enterococci
(VRE) isolates, respectively (23). Inducible genes encoding these
phenotypes alter cell wall synthesis and render strains resistant to
glycopeptides (22).

VanA and VanB types of resistance are primarily found among enterococci
isolated from clinical, veterinary, and food specimens (24), but not other
common intestinal or environmental bacteria. In the laboratory, however,
these genes can be transferred from enterococci to other bacteria (22). For
example, Staphylococcus aureus has been rendered vancomycin-resistant
through apparent transfer of resistance from E. faecalis on the surface of
membrane filters and on the skin of hairless obese mice (25), which
indicates that there is no biologic barrier to the emergence of
vancomycin-resistant S. aureus. Clinical isolates of highly
vancomycin-resistant S. aureus have yet to be identified, although strains
with reduced susceptibility to vancomycin have appeared (26). The mechanism
of resistance for these strains remains undetermined but does not appear to
involve genes associated with VanA or VanB phenotypes.

Epidemiology

Enterococci account for approximately 110,000 urinary tract infections,
25,000 cases of bacteremia, 40,000 wound infections, and 1,100 cases of
endocarditis annually in the United States (2,27,28). Most infections occur
in hospitals. Although several studies have suggested an increase in
nosocomial infection rates for enterococci in recent years, National
Nosocomial Infections Surveillance system data show little change in the
percentage of enterococcal bloodstream (12% vs. 7%), surgical site (15% vs.
11%), and urinary tract (14% vs. 14%) infections over the past 2 decades
(3,29). Adequate surveillance data prior to 1980 are not available.
Enterococcal infection deaths have also been difficult to ascertain because
severe comorbid illnesses are common; however, enterococcal sepsis is
implicated in 7% to 50% of fatal cases (6). Several case-control and
historical cohort studies show that death risk associated with
antibiotic-resistant enterococcal bacteremia is severalfold higher than
death risk associated with susceptible enterococcal bacteremia (30). This
trend will likely increase as MDR isolates become more prevalent.

Colonization and infection with MDR enterococci occur worldwide. Early
reports showed that in the United States, the percentage of nosocomial
infections caused by VRE increased more than 20-fold (from 0.3% to 7.9%)
between 1989 and 1993, indicating rapid dissemination. New database
technologies, such as The Surveillance Network (TSN) Database-USA, now
permit the assessment of resistance profiles according to species. TSN
Database electronically collects and compiles data daily from more than 100
U.S. clinical laboratories, identifies potential laboratory testing errors,
and detects emergence of resistance profiles and mechanisms that pose a
public health threat (e.g., vancomycin-resistant staphylococci).

Data collected by the TSN Database between 1995 and September 1, 1997
were analyzed to determine whether the earlier increase in vancomycin
resistance was unique to vancomycin, whether it represented a continuing
trend, and whether speciation is quantifiably important in analyzing
this trend. E. faecalis resistance to ampicillin and vancomycin is uncommon 
Figure 1); little change in resistance prevalence occurred from 1995 to 1997.
In contrast, E. faecium vancomycin and ampicillin resistance increased alarmingly. 
In 1997, 771 (52%) of 1,482 of E. faecium isolates exhibited vancomycin resistance,
and 1,220 (83%) of 1,474 isolates exhibited ampicillin resistance (Figure 1).
E. faecium resistance notwithstanding, E. faecalis remained by far the most 
commonly encountered of the two enterococcal species in TSN Database.
E. faecalis to E. faecium total isolates were approximately 4:1 (Figure 1),
blood isolates 3:1, and urine isolates 5:1. This observation underscores important
differences in the survival strategies and likelihood of therapeutic success, critical
factors usually obscured by lumping the organisms together as Enterococcus
species or enterococci. Widespread emergence and dissemination of
ampicillin and vancomycin resistance in E. faecalis would significantly
confound the current therapeutic dilemma. There is little reason to suspect
that vancomycin and ampicillin resistances only provide selective advantage
for the species faecium and not faecalis. The relative absence of these
resistances in E. faecalis may simply reflect a momentary lack of
penetrance and equilibration of the traits. Because of these important
differences between the two species, meaningful surveillance of
enterococcal resistance must include species identification.

[fig 1]
Figure 1. Epidemiology of enterococcal infection based on 15,203 susceptibility
results obtained by The Surveillance Network (TSN) Database-USA, 1995 to Sep 1, 1997. The 
increase in total numbers between 1995 and 1996 represents additional reporting centers
coming on line. Numbers for 1997 represent total collected for the partial year to Sep 1,
1997. 

Although exact modes of nosocomial transmission for MDR enterococci are
difficult to prove, molecular microbiologic and epidemiologic evidence
strongly suggest spread between patients, probably on the hands of
health-care providers or medical devices, and between hospitals by patients
with prolonged intestinal colonization. At least 16 outbreaks of MDR
enterococci have been reported since 1989 (31); all but two were due to E.
faecium. This disparity, particularly in view of the higher numbers of
clinical E. faecalis isolates, may reflect a reporting bias due to the
novelty of the combinations of resistance that occur in E. faecium. When
isolates from outbreaks of MDR enterococci have been analyzed by genetic
fingerprints, more than half involve clonally related isolates (18,32).

Prior treatment with antibiotics is common in nearly all patients colonized
or infected with MDR enterococci (33-35). Clindamycin, cephalosporin,
aztreonam, ciprofloxacin, aminoglycoside, and metronidazole use is equally
or more often associated with colonization or infection with MDR
enterococci than vancomycin use. Other risk factors include prolonged
hospitalization; high severity of illness score; intraabdominal surgery;
renal insufficiency; enteral tube feedings; and exposure to specific
hospital units, nurses, or contaminated objects and surfaces within
patient-care areas.

Infection Control

Controlling the spread of MDR enterococci among inpatients is difficult. We
know relatively little about the biology of enterococcal transmission or
the specific microbial factors favoring colonization by exogenous
enterococcal strains. Nevertheless, VRE infection control policies, which
could apply to MDR enterococci, were recently published by the Hospital
Infection Control Practices Advisory Committee (36). Control methods
include routine screening for vancomycin resistance among clinical
isolates, active surveillance for VRE in intensive care units, contact
isolation to minimize person-to-person transmission, and vancomycin
restriction.

These measures to limit VRE spread, however, have failed on occasion (35).
Not all hospitals can or are willing to perform active surveillance.
Because more patients are typically colonized with VRE (3% to 47%) than are
infected (35,37,38), and because intestinal colonization can be prolonged,
passive surveillance by routine cultures allows colonized inpatients to go
unidentified and serve as point sources for continued spread of VRE. Even
if all colonized inpatients are successfully identified, VRE may be spread
by health-care workers through either inadequate hand washing (39) or
through contact with items such as bedrails, sinks, faucets, and doorknobs
(enterococci can persist for weeks on environmental surfaces) (40).
Decontamination efforts must be rigorous.

The Hospital Infection Control Practices Advisory Committee strongly
recommended restricting oral and parenteral vancomycin to control VRE (36).
However, limiting use of vancomycin while ignoring widespread use of other
broad spectrum antibiotics likely will not lead to maximal control of VRE
or of MDR enterococci.

Antibiotics may promote colonization and infection with MDR enterococci by
at least two mechanisms. First, many broad spectrum antibiotics have little
or no anti-enterococcal activity, and administration commonly leads to
overgrowth of susceptible (or resistant) enterococci at sites at risk for
infection. Second, most antibiotics substantially reduce the normal
resistance of the intestinal tract to colonization by exogenous organisms
(41). Colonization resistance results primarily from the "limiting action"
of the normal anaerobic flora, and to a lesser extent from an intact
mucosa, gastric acid secretion, intestinal motility, and
intestinal-associated immunity (41). Antibiotic-induced alterations in the
protective flora of the intestine provide large footholds for colonization
with exogenous pathogens such as MDR enterococci (41). Antibiotic
restriction programs would be more effective if they included prudent
prescribing of all antibiotics, not just single agents such as vancomycin.
This approach substantially decreased intestinal colonization with VRE in
one hospital pharmacy that restricted vancomycin, cefotaxime, and
clindamycin (42).

At a minimum, a successful program for control of MDR enterococci requires
effective passive and active surveillance to identify colonized and
infected patients, absolute adherence to contact isolation by health-care
workers, rigorous decontamination of patient-contact areas and judicious
use or restriction of vancomycin and other broad spectrum antibiotics.

Therapeutic Approaches

Suitable antibiotics often are not available to treat MDR enterococcal
infections, e.g., endocarditis or bacteremia, in the presence of
neutropenia. Combinations of penicillin with vancomycin, ciprofloxacin with
ampicillin, or novobiocin with doxycycline, among others, have been used
(43) but can be unpredictable and remain clinically unproven. In one report
chloramphenicol successfully treated chloramphenicol-susceptible infections
(44), but these findings await confirmation in controlled trials.

Promising new antibiotics for MDR enterococcal infection under
investigation include fluoroquinolones, streptogramins, oxazolidinones,
semisynthetic glycopeptides, and glycylcyclines. Clinafloxacin, a
fluoroquinolone with improved potency against enterococci compared with
ciprofloxacin, has excellent activity against VRE, appears bactericidal in
vitro, and has been effective in treatment of enterococcal infections in a
murine model (45). Although single-step resistance to clinafloxacin could
not be detected in vitro, multistep resistance is readily achieved. Should
this agent gain approval for treatment of enterococcal infections,
selection for resistance may limit its effectiveness.

Quinupristin/dalfopristin (Synercid) is a combination of streptogramins A
and B that inhibits protein synthesis and has a narrower spectrum of
activity against enterococci than clinafloxacin (46). Many, but not all, E.
faecium isolates with VanA and VanB phenotypes are susceptible (47);
however, E. faecalis is uniformly resistant, and superinfection has been
reported during therapy (48). In addition, quinupristin/dalfopristin is
bacteriostatic only, potentially allowing emergence of resistance (49). For
these reasons the drug may have only a limited role in treating MDR
enterococcal infections. Novel oxazolidinones and glycylcyclines have also
shown potent activity against enterococci, including MDR enterococci
(50,51), but await further testing.

The substantial drawback of the broad spectrum approach is that the more
organisms affected (both protective commensals as well as pathogens), the
more opportunities for resistance to evolve. Broad spectrum antibiotics
permit empiric therapy in the absence of a specific diagnosis and generate
a more substantial return on investment in the short term. However, broad
spectrum antibiotics affect not only disease-causing organisms but also
commensals present in numbers large enough to generate resistance by
otherwise rare mutations or genetic exchange events. As long as market
forces favor development of broad spectrum therapeutics, a cycle of drug
introduction followed by emergence of resistance undoubtedly will continue.

Targeted Therapeutics

In contrast to the historical reliance on broad spectrum antibiotic
therapy, the continuing development and introduction of rapid diagnostic
techniques (52) may allow a more focused approach to infectious disease
therapy. Any of the myriad microbial-host interactions that subvert the
host response or damage tissues during an infection represent potential
therapeutic targets. However, many key interactions in disease pathogenesis
are specific to the organism involveda characteristic that is both a
strength and a weakness. Because of the specificity of these interactions,
rapid and accurate diagnosis is required. However, therapeutics aimed only
at interaction between host and a specific pathogen should leave the
diverse commensal flora essentially unaffected. As a result, the targeted
population would be restricted to the relatively small numbers of
disease-producing bacteria and would not likely reach the numbers or
diversity required to make development of resistance a statistical
probability.

The current spectrum of approaches to identify new antiinfective compounds
has two extremes: 1) screening vast libraries of compounds to identify
substances that by chance inhibit a microbial property and 2) detailed
study of interactions between host and parasite to identify critical events
leading to host tissue damage or compromise (53).

With a long-term view toward new therapeutic approaches as well as optimal
use of existing therapies, we and others have begun examining in detail the
interactions between enterococci and host (6). A major obstacle is that
enterococci also form part of the commensal or autochthonous flora; as
such, they are nearly devoid of traits traditionally associated with overt
pathogens and have subtle interactions with the host. Using inocula with as
few as 10 organisms, we have developed sensitive biologic systems for
examining the host-parasite interactions (54).

Although E. faecium strains are
resistant to vancomycin and
ampicillin more often than E.
faecalis strains, the relative
proportion of infections caused by
these species has not dramatically
changed in recent years (Figure 1).
Since both organisms are frequently
isolated from the commensal flora, this bias suggests that E. faecalis
traits confer a greater degree of intrinsic virulence, for example,
cytolysin production, pheromone-responsive plasmid transfer (and
accompanying production of aggregation substance), extracellular superoxide
production, and a newly identified surface protein tentatively termed Esp
(5,56,57) (Figure 2). These properties provide logical points of departure
for developing new targeted therapeutic approaches to enterococcal disease;
examination of more subtle interactions between E. faecium and host will
follow as an understanding of enterococcal biology evolves.

[fig 2]
Figure 2. Virulence traits and their association with enterococcal species.

Targeting the E. faecalis Cytolysin

Cytolysin is disproportionately expressed by E. faecalis strains associated
with disease (5,55,56). This cytolysin causes rupture of a variety of
target membranes, including bacterial cells, erythrocytes, and other
mammalian cells, with activity observed as a hemolytic zone on some types
of blood agar. Cytolysin contributes to the toxicity or lethality of
infection in several infection models and is associated with a fivefold
increased risk of sudden death from nosocomial bacteremia (54,56-59).
Cytolysin also contributes to the appearance of enterococci in a murine
bacteremia model (Figure 3; 45,60), an observation consistent with the
disproportionate representation of cytolytic strains among human blood
isolates (56,62).

[fig 3]
Figure 3. Cytolysin favors the appearance of circulating enterococci. In this
experiment, 10(sup)7 CFU of E. faecalis, either cytolytic FA2-2(pAM714) (60)
or noncytolytic FA2-2(pAM771) (64), were intraperitoneally injected (45) into groups of
five BalbC mice. Viable bacteria in liver, spleen, and the bloodstream were enumerated 48
hrs following injection, and significance assessed by Student's t-test. (P. Coburn, L.E.
Hancock, and M.S. Gilmore, in preparation).

Beginning with E.W. Todd in 1934 (63) and
culminating in a recent study (64), the
E. faecalis cytolysin has now been
characterized as a unique, extensively
modified bacterial toxin (Figure 4). The
cytolysin maturation pathway is ideally
designed for therapeutic targeting
because the two structural subunits are
activated by an extracellular protease,
an event that is accessible and
potentially inhibitable by a novel
therapeutic. Moreover, the activator
protease, CylA, belongs to the subtilisin
class of serine proteases (64), whose
structure-function relationships and
inhibitor design we are beginning to
understand. Investigations are in
progress to design and test inhibitors of
extracellular cytolysin activation to
determine whether a reduction by several
logs in the levels of circulating
enterococci can be attained as would be predicted
by the observed behavior
of cytolysin mutants (Figure 3).

[fig 4]
Figure 4. Cytolysin is expressed and processed through a complex maturation pathway
(64). The cytolysin precursors, CylL(sub L) and CylL(sub S), are ribosomally
synthesized. The putative modification protein, CylM, is required for the expression of
CylL(sub L) and CylL(sub S) in an activatable form, and the secreted forms, CylL(sub L)
and CylL(sub S) were recently shown to possess the amino acid lanthionine as the
result of posttranslational modification (64). CylL(sub L) and CylL(sub S) both
are secreted by CylB (65), which is accompanied by an initial proteolytic trimming event
(64) converting each to CylL(sub L)' and CylL(sub S)', respectively. Once
secreted, CylL(sub L)' and CylL(sub S)' are both functionally inactive until six
amino acids are removed from each amino terminus. This final step in maturation is
catalyzed by CylA (64), a subtilisin-type serine protease. Since this final catalytic
event is essential, occurs extracellularly, and is catalyzed by a class of enzyme for
which a substantial body of structural information exists, it represents an ideal
therapeutic target. As shown in Figure 3, inhibition of cytolysin by
mutation (or potentially by therapeutic intervention) results in a reduction by several
orders of magnitude in the number of circulating organisms. 

An inhibitor of cytolysin activation,
accompanied by appropriate rapid
diagnostics, would be of potential
value in treating bacteremia caused
by cytolytic strains of E. faecalis
without affecting commensal flora.
Development of resistance should be
exceedingly improbable because of the
small number of bacteria targeted and
because unlike antibiotics, cytolysin
inhibitors would not act directly on
the organism itself.

Other Enterococcal Targets

Several laboratories are using
information on the E. faecalis genome
and genomes of other pathogens to
identify therapeutic targets (66) and
facilitate studies on pathogenesis
for the remaining 60% of noncytolytic
enterococcal infections. The genome
of an E. faecalis strain that caused
multiple hospital infections (56) was
sampled at high frequency by sequence
analysis. Several sequences appeared
to have a role in host-parasite
interaction. The gene specifying Esp
encodes an apparent surface protein
of unusual repeating structure (67).
Although a role for this protein in
enterococcal infection has yet to be
determined, its distribution among
clinical and commensal strains is
tantalizing: 29 of 30 strains with
this gene were recovered from
patients with bacteremia or
endocarditis; one of 34 isolates
obtained from healthy volunteers
possessed Esp. The core of this large
protein (inferred mass of 202 kDa)
consists of a series of 82 amino acid
repeats encoded by highly conserved
tandem 246 base pair repeats. Lack of
divergence in repeat sequences suggests that recombination occurs at high
frequency, perhaps during infection. Moreover, the number of repeats
observed in homologous genes from different E. faecalis isolates is 3 to 9
(67). This gene is flanked by a sequence similar to the transposase of
IS905. None of 24 clinical or laboratory E. faecium isolates had this gene
(67; V. Shankar, G. Lindahl, and M. Gilmore, unpub. data).

A second promising lead involves a series of genes encoding products highly
related to enzymes involved in O-antigen synthesis in gram-negative
bacteria (68). Preliminary evidence suggests that in E. faecalis these
genes contribute to cell wall carbohydrate synthesis and that this
carbohydrate relates to persistence in vivo. A knockout in one of these
genes results in a strain with normal in vitro growth, but after
subcutaneous injection, the mutant was more readily cleared than the wild
type parental strain (68). One of the genes studied was present in all E.
faecalis strains examined, whereas another occurs only in E. faecalis
strains that share a periodate-susceptible epitope (68). Collectively,
these data indicate that enzymes for synthesis of E. faecalis surface
carbohydrates are important for persistence in vivo and may represent a
useful therapeutic target. Taking a different approach, Arduino et al.
(69,70) identified a protease-resistant, periodate susceptible substance
associated with some strains of E. faecium, but not E. faecalis, which
conferred resistane to phagocytosis in vitro. The relationship between the
putative carbohydrate of E. faecalis under study above and the inhibitory
substance of E. faecium remains to be determined. It may be found that many
enterococci produce such carbohydrates at biologically significant levels
in vivo, but only some strains of E. faecium do so in vitro.

Finally, recent observations indicate that nearly all E. faecalis strains,
and only a few E. faecium strains, generate substantial extracellular
superoxide. When E. faecalis isolates from patients with endocarditis and
bacteremia were compared with isolates from healthy volunteers (71), on
average, extracellular superoxide production was 60% higher among blood
isolates than commensal strains. These data raised several questions: Do E.
faecalis that produce larger amounts of extracellular superoxide possess
greater metabolic flexibility, facilitating adaptation to nonintestinal
infection sites? Does free radical production lead to host cell damage,
allowing release of normally sequestered nutrients (e.g., hemin) that might
promote enhanced E. faecalis growth through cytochrome formation? Might
antioxidants modulate colonization or invasive infection? Answers to these
questions may provide new insights into the transition from intestinal
colonization to infection and may suggest new preventive strategies.

Obstacles to Further Development

Although important insights into enterococcal biology and pathogenesis are
being gleaned from a reverse genetic approach, a paucity of information
still exists on how enterococci colonize the intestinal tract and cause
infection. For example, do E. faecalis or E. faecium colonize the colon
through specific interactions with ligands on human epithelial cells or
intestinal mucin? Do MDR enterococci possess alternate binding activities
that enable them to colonize the intestinal tract at new sites without
competing with the indigenous enterococci? Do probiotics have a role in
restoring colonization resistance to an intestinal ecology altered by broad
spectrum antibiotics?

Is enough being done to combat the emergence of highly resistant nosocomial
pathogens? To effectively compete, industry remains highly responsive to
market opportunities. Research in the public sector has been slow to
respond, and as a result, our understanding of the biology of enterococcal
infection is inadequate. Reasons for the modest public sector response
include the following. 1) The emergence of resistant enterococci coincided
with a reduction of public support for non-AIDS related infectious disease
research. 2) The pathogenesis of nosocomial infection deviates from
paradigms established for obligate pathogens. 3) The research
infrastructure is relatively small because of the low importance
traditionally attached to enterococci as etiologic agents of human disease
and the deemphasis on antibiotic resistance research in the 1980s.

Conclusions

Historically, substantial resources have been invested in developing an
in-depth understanding of the molecular biology of model organisms. During
the 1960s and 1970s, when gram-negative organisms were leading causes of
hospital- and community-acquired infections and gram-positive organisms
were typically sensitive to existing antibiotics (72), a sizable investment
in gram-negative model organisms was appropriate. However, with the
emergence of gram-positive organisms as leading causes of both hospital-
and community-acquired infection in the 1990s, a reevaluation of public
research priorities is warranted.

Since antibiotic use became widespread 50 years ago, bacteria have steadily
and routinely developed resistance. Control of the emergence of resistance
will depend on new approaches to prudent antibiotic use in hospitals and
clinics, based in part on improved surveillance for MDR enterococci and on
better systems to encourage staff adherence to contact isolation
procedures. Equally important will be development of new drugs with
narrower spectra of activity aimed at known and potentially new targets and
the evolution of market conditions that favor their use.

Portions of the work described were supported by Veterans Administration
Merit Review Program, grants from the Public Health Service (EY08289 and
AI41108), and an unrestricted award from Research to Prevent Blindness,
Inc.

Dr. Huycke is an associate professor in the Infectious Diseases Section,
Department of Medicine, Oklahoma University Health Sciences Center. He is
interested in enterococcal pathogenesis as it relates to extracellular
superoxide production by E. faecalis.

Address for correspondence: Michael Gilmore, Department of Microbiology and
Immunology, University of Oklahoma Health Sciences Center, PO Box 26901,
Oklahoma City, OK 73190, USA; fax: 405-271-8128; e-mail:
mgilmore@aardvark.ouknor.edu.

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