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Enterococci have been known for over a century to be
capable of causing infections in humans 1, 2. For a long time, these
infections were limited in numbers and mostly caused by Enterococcus faecalis.
In the last decades enterococci have emerged as important nosocomial pathogens,
largely due to their intrinsic antimicrobial resistance and their vast capacity
to acquire antimicrobial resistance 3, 4. Their genomic plasticity has also
contributed to their adaptation to the hospital environment. In the early 1980s
E. faecalis accounted for 90% of enterococcal 
infections 5. Subsequently, ampicillin resistant Enterococcus faecium
started to emerge 6, and in 1986 transferable high-level vancomycin resistant
enterococci (VRE) was discovered 7, 8. In addition, E. faecium was shown to
easily acquire resistance to other antimicrobials 9. Since then, a gradual
increase in enterococcal infections has been seen. E. faecium infections have
increased relative to E. faecalis and have partially replaced E. faecalis as a
cause of hospital associated infections. Now the prevalence of infections
caused by E. faecium is close to that of E. faecalis 10-12. E. faecalis have
also been shown to acquire antimicrobial resistance, high-level gentamicin
resistance (HLGR) in particular, but resistance to ampicillin and vancomycin is
infrequent 13. Enterococcal infections are now the 3rd and 4th most frequent
microorganism isolated from hospital associated infections in the US and
Europe, respectively 10, 14. 

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Enterococci are commensals of the human and animal
intestinal flora 15-17. They are also commonly used in food fermentation
18-20 and easily detectable in environmental sources such as in water, plants
and soil 21-23. Until the 1980s, species that today belong to the
Enterococcus genus were classified as streptococci. In 1984 DNA homology
studies showed that  Streptococcus  faecalis and Streptococcus  faecium were so distantly related to
streptococci that they were transferred to another genus; Enterococcus faecalis
and Enterococcus faecium, respectively 24. In the beginning of the 19th
century, S. faecalis and S. faecium were considered the same species 1, but
during the 1940s and 1950 studies showed that the two organisms had different
biochemical characteristics and by the mid1960s they were accepted as two
distinct species 25. A number of other enterococci have been isolated 5,
26, and by 01.02.2012 there were 47 species in the Enterococcus genus
registered in the Taxonomy browser in GenBank
(  Enterococci belong to the phylum Firmicutes
and the family of Enterococcacae. They are Gram positive facultative anaerobic
organisms that are catalase negative, with the ability to hydrolyse esculin in
the presence of bile. They can grow under harsh conditions, including both 10°C
and 45°C , in the presence of 6,5% NaCl, and at pH 9,6. In addition,
enterococci survive for 30 minutes at 60°C 27. The GC-content in the
enterococci is low (36-40%), but can vary within the genome 28-30. Sequencing
of E. faecium and E. faecalis genomes have shown that both have an open pan
genome, which means there is no limit to the number of genes that can be part
of the joint genome of all bacteria within the species. It also revealed that
the genomes are very flexible, with a large ability to recombine, that are at
least in part due to the high numbers of 
IS- and other mobile genetic elements present in these genomes 28, 29,





Hospital acquired infections (HAI) are described as
an infection occurring during hospitalization. Definition criteria often
include that the infection was neither present nor incubating at the time of
hospital admission. As a consequence, in many epidemiological surveillance
systems, these infections are required to appear no earlier than 48 hours after
hospital admission to be defined as HAI 14, 35, 36. The European Centre for
Disease prevention and Control (ECDC) have estimated the prevalence of HAI in
European acute care hospitals to range from 3,5%-10,5% with an average of 7,1%
among admitted patients. From this prevalence, the cumulative incidence have
been estimated to approximately 5,1% 14. This means that for every 100
persons who are admitted to the hospital, 5 persons will get a hospital
acquired infection. The economic burden of HAI is a comprehensive and complex
calculation, and the transferability between different studies have proven low
37. To give an idea of the increased cost attributable directly to HAI, we
can calculate the cost of the lengthened hospital stay as a result of HAI. A
prudent valuation has estimated that HAI lengthen the hospital stay with an
average of 4 days 38.  The average
hospital stay has been calculated to cost EUR 435 per day 39, which means
that for every 100 persons admitted to the hospital, HAI will increase the
costs with EUR 8700. This is only estimating the direct cost of the lengthen
stay, not considering any indirect costs such as cost related to the need for
additional medical procedures, the need for isolation, loss of income,
increased morbidity or increased mortality. The share of deaths contributable
to HAI is substantial. The US CDC estimated the direct attributable mortality
of HAI to be 0,9%, in addition it contributed to 2,7% of deaths 38. Combined
with antimicrobial resistance, the consequences of HAI are even greater: higher
costs, more morbidity and more mortality. Carmeli et al. shoved that for VRE
infections the multiplicative effect for lengthened hospital stay was 1,73 and
for hospital cost 1,4. Morbidity was also significantly increased and the risk
of death was doubled 40.    




Enterococci are a common cause of HAI worldwide. In
Europe, the prevalence of enterococcal HAI is around 8%, and enterococci are
only outnumbered by Escherichia coli, Staphylococcus aureus and Pseudomonas
aeruginosa 14. Although enterococci do not reach the top-ten list of nosocomial
outbreak pathogens 41-43, ECDC has placed them on the list of pathogens
posing a major threat to healthcare systems 14. This is in large part a
result of the increasing antimicrobial resistance in enterococci. In the US,
80% of E. faecium isolates are vancomycin resistant 10. In Europe the
prevalence of VRE has traditionally been low, and in the Scandinavian countries
prevalence is still below 1%. However, increasing rates of VRE have been
reported from many European countries, and in Greece and Ireland the prevalence
is even >30% 44. In Norway, as in the rest of the world, the prevalence of
enterococcal infections is increasing, and E. faecium isolated from blood
cultures have increased nearly a 4 –fold, while the number of E. faecalis isolates
have doubled (Figure 1). The success of E. faecium has been tributed to the
success of hospital adapted lineages of this species (see later). In Norway
enterococci are the 5th most common aetiological agent causing bacteraemia
45. In parallel to the increase in enterococcal infections in Norway, an
increase of high-level gentamicin resistance (HLGR) have been observed (Figure
2;45, 46. This seems to be part of an international trend occurring in both
European, Asian and South American countries 47-53.

Enterococci are considered opportunistic pathogens.
As commensals of the human gut flora they do not normally cause infections in
healthy people, with the exception of occasional urinary tract infections.  However, enterococci have proven very competent
in causing opportunistic infections in hospitalized patients, particularly in
debilitated hosts 54-57. Several studies have shown that exposure to
antimicrobials facilitates changes in the intestinal microbiota, which promote
colonisation by enterococci 58-62. It has also been shown that increased
density of colonizing enterococci in the intestine precedes bloodstream
infections 62. Other risk factors for colonization and subsequent infections
with enterococci include admission to a critical care unit, co-morbidity,
exposure to other patients with hospital adapted enterococci, long period of
hospitalization, haemodialysis and solid organ and bone marrow transplantation
40, 63-67. Most studies investigating risk factors focus on vancomycin resistant
enterococci, but the crucial determinant giving enterococci the ability to
colonize and infect a host is not only vancomycin resistance. Hence one could
assume the risk factors for acquiring enterococcal infection should be somewhat
similar between vancomycin resistant (VR) and vancomycin susceptible
enterococci (VRE).

Enterococci can cause a variety of infections, most
of them facilitated by indwelling devices or structural anatomic abnormalities.
Urinary tract infections (UTI) are the most common enterococcal infection, and
often associated with urinary catheters 68. If not accompanied by
bacteraemia, it generally only requires single-drug therapy, although seriously
ill patients with pyelonephritis may benefit from combination therapy 68, 69.
Intra-abdominal and pelvic infections are often polymicrobial in origin.
Although enterococci are detected in 20% of these 70, it is debatable to what
extent they contribute to the infections 71. However, these infections are
common sources of bacteraemia 72, 73, hence antimicrobial therapy active
against enterococci is regularly recommended 70. Bacteraemia is not
necessarily accompanied by an infection, but is none the less a bacterial
invasion of the body. The source of the bacteraemia is often an infection or an
indwelling device, but translocation of enterococci across intact intestinal
epithelial cells may also lead to bacteraemia 72, 74. The percentage of
patients were endocarditis is the cause of enterococcal bacteraemia varies from
about 1% to 32% in different studies 75. Enterococci account for 5-20% of
cases of endocarditis and are thus the 2nd -3rd most common cause of
endocarditis. Enterococcal meningitis is rare accounting for about 0.3% to 4%
of meningitis cases 76, 77. Severe enterococcal infection generally requires
combination therapy 75, 78-80.



Enterococci are traditionally treated with a
combination of cell wall active antimicrobials such as ?-lactams or
glycopeptides, and aminoglycosides 80. However, the increased rates of
?lactam and glycopeptide resistance in E. faecium and aminoglycoside resistance
in both E. faecium and E. faecalis have called for the use of other and perhaps
less efficient drugs.  

Aminoglycoside antibiotics were one of the early
discovered antibiotics and have been in use for over 60 years. They bind to the
30S ribosomal subunit, which plays a crucial role in providing high-fidelity
translation of genetic material 81, rendering the ribosome unavailable for
translation and thereby resulting in cell death 82. Aminoglycosides have a
broad antimicrobial spectrum covering a wide variety of aerobe Gram negatives
and some Gram positives 83. They display concentration-dependent bactericidal
activity and is effective even when the bacterial inoculum is large 84. The
aminoglycosides are seldom drugs of first choice for monotherapy of infections,
except for some cases of uncomplicated urinary tract infections 85. Because
of their synergism with cell wall synthesis inhibitors, they are recommended as
part of an empirical combination therapy for severe infections such as
septicaemia, nosocomial respiratory tract infections, complicated
intra-abdominal infections and enterococcal endocarditis 80, 86-93. Synergism
presumably arises as the result of enhanced intracellular uptake of
aminoglycosides caused by the increased permeability of bacteria after
incubation with cell wall synthesis inhibitors such as ?-lactams and
glycopeptides 91, 94, 95. Resistance rarely develops during the course of
treatment 96, 97. Gentamicin is the aminoglycoside most often used, because
of its low cost and reliable activity against Gram negative aerobes 98. The
major limitations of aminoglycosides is a relatively low therapeutic index with
both nephrotoxicity and ototoxicity, and that they are not absorbed orally due
to their cationic nature and thus must be given parentally by either an
intravenous or intramuscular route 96, 98.  

Cell wall active antimicrobials such as ?-lactams
and glycopeptides act by inhibiting the synthesis of the peptidoglycan layer of
bacterial cell walls 99, 100. Penicillins are considered bacteriostatic
against enterococci, and are the most widely used antimicrobials in the world
101. Glycopeptides only work on Gram positive bacteria and is considered
bacteriostatic against enterococci 3, 102. In the last decade several
antimicrobials with effect on enterococci have emerged. They all exhibit less
than 100% clinical and microbiological success, usually around 70% 80, 103.
To improve their efficacy and reduce the development of resistance, it is
preferable to employ them as part of a combination regimen 80, 103. Linezolid
inhibits protein synthesis and is active against all clinically important Gram
positive bacteria, although it only displays a bacteriostatic effect 104,
105. Daptomycin interferes with the cytoplasmic membrane causing
depolarization and cessation of protein-, DNA and RNA-synthesis 106, 107. It
has concentration-dependent bactericidal activity against enterococci 108,
109. Quinupristin-dalfopristin (Q/D) is a streptogramin antibiotic that is
only active against E. faecium. It inhibits protein synthesis and is considered
bacteriostatic against enterococci 110. Tigecyclin is a broad-spectrum
antibiotic that inhibits the protein synthesis. A recent review showed that it
was more effective against enterococci than other Gram positive bacteria, but
infections included were mostly skin and soft tissue infections and intra-abdominal
infections 111, 112.



The discovery of antibiotics is considered one of
the most significant health related events of modern times and antibiotic
therapy is one of the cornerstones in modern medicine. Use and misuse of
antimicrobials in human medicine and animal husbandry over the past 70 years
have caused an unremitting selection pressure that has given rise to
innumerable microorganisms resistant to these medicines. The use of
antimicrobials are positively correlated to the emergence of resistant bacteria
113, 114. Several bacteria in the hospital setting in many countries
worldwide are now multiresistant 10, 14, leaving few treatment options.
Hence, the development of antimicrobial resistance by bacteria constitutes a
major threat to human health (WHO)




Intrinsic resistance is a species characteristic,
and thus present in all members of the species. 
Enterococci are resistant to most ?-lactam antibiotics due to a
penicillin-binding protein (PBP) that has a low affinity for beta-lactam agents
115, 116.  For ampicillin,
ureidopenicillins, penicillin and imipenem the resistance is only low level. E.
faecium  generally display higher MICs
than E. faecalis 5. Enterococci display low level resistance to
aminoglycosides (se later) and lincosamides 5. E. faecalis also possesses an
efflux pump conferring resistance to lincosamides and dalfopristin 117. In
addition, many wild-type enterococci possess endogenous efflux pumps that
excrete chloramphenicol  making them low level
resistant 118. Most enterococci are susceptible to co-trimoxazole in vitro,
but this combination does not work in vivo, because enterococci are able to
incorporate exogenous folic acid which enables them to bypass the inhibition of
folate synthesis caused by cotrimoxazole 5. 



A diversity of antimicrobial resistance genes have
been demonstrated in the human gut microflora 119. As inhabitants of the
human intestinal flora, enterococci are in a position to acquire resistance
genes from this  community, thus making
them notoriously difficult to treat and enabling them to transfer resistance
genes to even more pathogenic bacteria, such as vanA to S. aureus 120, 121.





  In the last
two decades, E. faecium have evolved as a common nosocomial pathogen,
increasing the total burden of enterococcal infections and partially replacing
E. faecalis as a cause of HAI 11. In the beginning of the millennium, genotypic
population studies 122, 123 showed distinct genetic lineages spreading in the
hospital, suggesting the existence of a specific subpopulation of E. faecium
associated with hospital acquired infections, different from the community and
animal population. Ampicillin resistance and esp (enterococcal surface protein-
a putative virulence gene) were the early markers associated with this
subpopulation 146, 148. Later a pathogenicity island (PAI) containing esp
124, IS16 29, 125 and quinolone resistance was also linked to these strains
126, 127. In addition, putative virulence genes 128, 129, and several
surface proteins are enriched in this hospital associated subpopulation 130,
131. A large genotypic study of population structure, typing over 400 isolates
by Multi locus sequence typing (MLST) and analyzing it with eBURST, confirmed
the existence of a subpopulation of E. faecium representing clinical and
hospital outbreak strains 132. It demonstrated genetic clustering of hospital
associated strains, named clonal complex 17 (CC17) that was strongly associated
with ampicillin resistance and the esp containing PAI. ST17 was presumed to be
the founder of this clonal complex.  A
microarraybased comparative genomic hybridization of mixed whole genomes,
hybridized against 97 isolates also supported the presence of a distinct
phylogenetic group of hospital associated strains 29. Many publications
worldwide have acknowledged CC17 as by far the most prevalent genetic
subcluster causing hospital acquired infections 51, 128, 133-135. The seven
major hospital associated STs (ST16, ST17, ST18, ST78, ST192, ST202 and ST203)
accounts for 56% of the hospital associated isolates 136. Later it has been
reported that eBURST based clustering of MLST data to determine evolutionary
decent is inaccurate in species with high levels of recombination such as E.
faecium 137. By using other approaches such as ClonalFrame 138 based
phylogenetic trees, constructed from the concatenation of the seven MLST
housekeeping genes 139, or a Bayesian modeling approach using BAPS software 161,
165, it has been showed that the CC17 subpopulation has not recently evolved
from a single common ancestor; the hospital associated subpopulation  is not clonal (ST17 is not the founder), but
rather polyclonal. This polyclonal subpopulation constitutes several lineages
that seem to have co-evolved into the clade now 
commonly known as hospital associated E. faecium. A recent study that
inferred phylogeny from 21 publically available E. faecium genomes by aligning
100 orthologs, showed a distinct separation of community-associated and
hospital associated strains. They estimated the two lineages to have diverged
over 300 000 years ago 33.  The
hospital adapted subpopulation of E. faecium seems to have exploited a novel
ecological niche- the hospital setting. They seem to be less fit when living
outside the healthcare boundaries as the seven major hospital associated STs
(ST16, ST17, ST18, ST78, ST192, ST202 and ST203) are only sporadically (41/513)
found among non-hospital isolates 136. This type of niche-exploitation often
starts with adaptive changes 141. Exactly which traits have given these
strains the upper hand in the hospital setting is not known, but several
properties have been suggested. Ampicillin resistance is one of the markers
strongest associated with this subpopulation, thus it is suggested that the
acquisition of ampicillin resistance was one of the vital traits enabling the
strains to enter the hospitals and evolve into successful nosocomial pathogens
125.  This type of adaptive change may
give rise to an amplifying selective process where isolates with the adaptive
change (e.g. ampicillin resistance), more easily can acquire additional
adaptive changes (e.g. changes in metabolism and other cellular processes)
improving their relative fitness 142, 144. The flexibility of the E. faecium
genome is believed to significantly contribute to the hospital adaptation.
Mobile genetic elements (MGE), particularly IS elements are believed to
increase the genome plasticity and facilitate adaptive changes, thus enhancing
the genetic variability in the hospital adapted strains 29, 32. In the last
years it has become apparent that megaplasmids are abundant among E. faecium,
suggesting they play a role in the adaptation of E. faecium to particular hosts
49,. Considering that megaplasmids had not been recognized among enterococci
before the 1990s 145, and have been shown to play a role in both
colonization, virulence and resistance in hospital associated E. faecium (se
later) 145 they may have played an important role in the recent success of
these strains.  


The available data indicates that E. faecalis has an
epidemic population structure dominated by a limited number of genetic lineages
with an overrepresentation of clonal complexes CC2, CC9, CC10, CC16, CC21,
CC30,CC40 and CC87 13, 176-178. CC2, CC9 and CC87 are considered high risk
CCs, as they are enriched in multidrug resistant isolates causing infections in
hospitalized patients 13, 168, 176. CC2 is a globally dispersed hospital
associated lineage highly capable of causing infections 13, 178-180. Solheim
et al. showed that over 250 genes were significantly enriched in CC2 isolates.
Most of these genes have not been characterized, but some genes were shown to
be located within mobile elements such as phage03, a putative integrative
conjugative element and a vanB associated genomic island 142. CC87 is
particularly dominating in Poland 181, but are also found in other European
countries as well as in the US 13. CC9 is spread globally, but high rates
have especially been reported in Spain 17.  

The seven most prevalent STs among clinical and
outbreak-associated E. faecalis (ST6, ST9, ST16, ST21, ST28 ST40 and ST87),
account for only 37% of the hospital associated isolates 136. In contrast
this is 56% for the seven most prevalent hospital associated E. faecium STs.
Some E. faecalis STs (ST16, ST21, ST28 and ST40) are also found frequently in
the community, including farm animals and food products isolates 13,, indicative
of a reduced host specificity. It has been shown that near 60% of patients
diagnosed with Vancomycin resistant (VR) E. faecalis bacteraemia in an US
hospital, where infected prior to hospitalisation, and that bacteraemia caused
by VR E. faecalis was significantly more likely to be present on admission than
bacteraemia caused by VR E. faecium . A recent study showed that CC21, CC16 and
CC40 showed better in vitro fitness than those linked to nosocomial infections
(CC2, CC9, CC87)  This indicates that hospital
associated CCs have acquired genetic elements, encoding specific traits
(antibiotic resistance, virulence genes) making them successful in the hospital
environment, but less fit in the environment. The most recent study on E.
faecalis population structure of human isolates 13 showed that CC2 and CC87
were found exclusively in hospitals. It also showed that the six most commonly
detected CCs (CC2, CC16, CC21, CC30, CC40 and CC87) accounted for 57% of the
hospital isolates. Comparison of gene tree topologies of individual MLST genes
indicates that recombination rates in E. faecalis are even higher than in E.
faecium. Hence, recombination seems to be the driving force in diversification
and evolution of this species 143. Thus it is may be more accurate to
consider CCs rather than STs as genetic lineages in E. faecalis.  






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