INTRODUCTION  Enterococci have been known for over a century to becapable of causing infections in humans 1, 2. For a long time, theseinfections 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 capacityto acquire antimicrobial resistance 3, 4. Their genomic plasticity has alsocontributed to their adaptation to the hospital environment. In the early 1980sE. faecalis accounted for 90% of enterococcal infections 5. Subsequently, ampicillin resistant Enterococcus faeciumstarted to emerge 6, and in 1986 transferable high-level vancomycin resistantenterococci (VRE) was discovered 7, 8. In addition, E. faecium was shown toeasily acquire resistance to other antimicrobials 9.

Since then, a gradualincrease in enterococcal infections has been seen. E. faecium infections haveincreased relative to E. faecalis and have partially replaced E. faecalis as acause of hospital associated infections.

Now the prevalence of infectionscaused by E. faecium is close to that of E. faecalis 10-12. E.

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faecalis havealso been shown to acquire antimicrobial resistance, high-level gentamicinresistance (HLGR) in particular, but resistance to ampicillin and vancomycin isinfrequent 13. Enterococcal infections are now the 3rd and 4th most frequentmicroorganism isolated from hospital associated infections in the US andEurope, respectively 10, 14.     GENERAL CHARACTERISTICS Enterococci are commensals of the human and animalintestinal flora 15-17. They are also commonly used in food fermentation18-20 and easily detectable in environmental sources such as in water, plantsand soil 21-23. Until the 1980s, species that today belong to theEnterococcus genus were classified as streptococci. In 1984 DNA homologystudies showed that  Streptococcus  faecalis and Streptococcus  faecium were so distantly related tostreptococci that they were transferred to another genus; Enterococcus faecalisand Enterococcus faecium, respectively 24. In the beginning of the 19thcentury, S. faecalis and S.

faecium were considered the same species 1, butduring the 1940s and 1950 studies showed that the two organisms had differentbiochemical characteristics and by the mid1960s they were accepted as twodistinct species 25. A number of other enterococci have been isolated 5,26, and by 01.02.

2012 there were 47 species in the Enterococcus genusregistered in the Taxonomy browser in GenBank(http://www.ncbi.nlm.nih.gov/taxonomy/?term=enterococcus).

  Enterococci belong to the phylum Firmicutesand the family of Enterococcacae. They are Gram positive facultative anaerobicorganisms that are catalase negative, with the ability to hydrolyse esculin inthe presence of bile. They can grow under harsh conditions, including both 10°Cand 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 theenterococci is low (36-40%), but can vary within the genome 28-30.

Sequencingof E. faecium and E. faecalis genomes have shown that both have an open pangenome, which means there is no limit to the number of genes that can be partof the joint genome of all bacteria within the species. It also revealed thatthe genomes are very flexible, with a large ability to recombine, that are atleast in part due to the high numbers of IS- and other mobile genetic elements present in these genomes 28, 29,31-34.

   CLINICALSIGNIFICANCE OF ENTEROCOCCI HOSPITAL ASSOCIATED INFECTIONHospital acquired infections (HAI) are described asan infection occurring during hospitalization. Definition criteria ofteninclude that the infection was neither present nor incubating at the time ofhospital admission. As a consequence, in many epidemiological surveillancesystems, these infections are required to appear no earlier than 48 hours afterhospital admission to be defined as HAI 14, 35, 36. The European Centre forDisease prevention and Control (ECDC) have estimated the prevalence of HAI inEuropean 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 havebeen estimated to approximately 5,1% 14. This means that for every 100persons who are admitted to the hospital, 5 persons will get a hospitalacquired infection. The economic burden of HAI is a comprehensive and complexcalculation, and the transferability between different studies have proven low37.

To give an idea of the increased cost attributable directly to HAI, wecan calculate the cost of the lengthened hospital stay as a result of HAI. Aprudent valuation has estimated that HAI lengthen the hospital stay with anaverage of 4 days 38.  The averagehospital stay has been calculated to cost EUR 435 per day 39, which meansthat for every 100 persons admitted to the hospital, HAI will increase thecosts with EUR 8700. This is only estimating the direct cost of the lengthenstay, not considering any indirect costs such as cost related to the need foradditional medical procedures, the need for isolation, loss of income,increased morbidity or increased mortality. The share of deaths contributableto HAI is substantial. The US CDC estimated the direct attributable mortalityof HAI to be 0,9%, in addition it contributed to 2,7% of deaths 38. Combinedwith antimicrobial resistance, the consequences of HAI are even greater: highercosts, more morbidity and more mortality.

Carmeli et al. shoved that for VREinfections the multiplicative effect for lengthened hospital stay was 1,73 andfor hospital cost 1,4. Morbidity was also significantly increased and the riskof death was doubled 40.             EPIDEMIOLOGY Enterococci are a common cause of HAI worldwide. InEurope, the prevalence of enterococcal HAI is around 8%, and enterococci areonly outnumbered by Escherichia coli, Staphylococcus aureus and Pseudomonasaeruginosa 14.

Although enterococci do not reach the top-ten list of nosocomialoutbreak pathogens 41-43, ECDC has placed them on the list of pathogensposing a major threat to healthcare systems 14. This is in large part aresult of the increasing antimicrobial resistance in enterococci. In the US,80% of E. faecium isolates are vancomycin resistant 10. In Europe theprevalence of VRE has traditionally been low, and in the Scandinavian countriesprevalence is still below 1%. However, increasing rates of VRE have beenreported from many European countries, and in Greece and Ireland the prevalenceis even >30% 44.

In Norway, as in the rest of the world, the prevalence ofenterococcal infections is increasing, and E. faecium isolated from bloodcultures have increased nearly a 4 –fold, while the number of E. faecalis isolateshave doubled (Figure 1).

The success of E. faecium has been tributed to thesuccess of hospital adapted lineages of this species (see later). In Norwayenterococci are the 5th most common aetiological agent causing bacteraemia45. In parallel to the increase in enterococcal infections in Norway, anincrease of high-level gentamicin resistance (HLGR) have been observed (Figure2;45, 46.

This seems to be part of an international trend occurring in bothEuropean, 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 inhealthy people, with the exception of occasional urinary tract infections.

  However, enterococci have proven very competentin causing opportunistic infections in hospitalized patients, particularly indebilitated hosts 54-57. Several studies have shown that exposure toantimicrobials facilitates changes in the intestinal microbiota, which promotecolonisation by enterococci 58-62. It has also been shown that increaseddensity of colonizing enterococci in the intestine precedes bloodstreaminfections 62. Other risk factors for colonization and subsequent infectionswith enterococci include admission to a critical care unit, co-morbidity,exposure to other patients with hospital adapted enterococci, long period ofhospitalization, haemodialysis and solid organ and bone marrow transplantation40, 63-67. Most studies investigating risk factors focus on vancomycin resistantenterococci, but the crucial determinant giving enterococci the ability tocolonize and infect a host is not only vancomycin resistance.

Hence one couldassume the risk factors for acquiring enterococcal infection should be somewhatsimilar between vancomycin resistant (VR) and vancomycin susceptibleenterococci (VRE).Enterococci can cause a variety of infections, mostof them facilitated by indwelling devices or structural anatomic abnormalities.Urinary tract infections (UTI) are the most common enterococcal infection, andoften associated with urinary catheters 68. If not accompanied bybacteraemia, it generally only requires single-drug therapy, although seriouslyill 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 whatextent they contribute to the infections 71. However, these infections arecommon sources of bacteraemia 72, 73, hence antimicrobial therapy activeagainst enterococci is regularly recommended 70.

Bacteraemia is notnecessarily accompanied by an infection, but is none the less a bacterialinvasion of the body. The source of the bacteraemia is often an infection or anindwelling device, but translocation of enterococci across intact intestinalepithelial cells may also lead to bacteraemia 72, 74. The percentage ofpatients were endocarditis is the cause of enterococcal bacteraemia varies fromabout 1% to 32% in different studies 75. Enterococci account for 5-20% ofcases of endocarditis and are thus the 2nd -3rd most common cause ofendocarditis. Enterococcal meningitis is rare accounting for about 0.3% to 4%of meningitis cases 76, 77. Severe enterococcal infection generally requirescombination therapy 75, 78-80.      ANTIMICROBIAL USED TO TREAT ENTEROCOCCAL INFECTIONEnterococci are traditionally treated with acombination of cell wall active antimicrobials such as ?-lactams orglycopeptides, and aminoglycosides 80.

However, the increased rates of?lactam and glycopeptide resistance in E. faecium and aminoglycoside resistancein both E. faecium and E. faecalis have called for the use of other and perhapsless efficient drugs.   Aminoglycoside antibiotics were one of the earlydiscovered antibiotics and have been in use for over 60 years.

They bind to the30S ribosomal subunit, which plays a crucial role in providing high-fidelitytranslation of genetic material 81, rendering the ribosome unavailable fortranslation and thereby resulting in cell death 82. Aminoglycosides have abroad antimicrobial spectrum covering a wide variety of aerobe Gram negativesand some Gram positives 83. They display concentration-dependent bactericidalactivity and is effective even when the bacterial inoculum is large 84. Theaminoglycosides are seldom drugs of first choice for monotherapy of infections,except for some cases of uncomplicated urinary tract infections 85. Becauseof their synergism with cell wall synthesis inhibitors, they are recommended aspart of an empirical combination therapy for severe infections such assepticaemia, nosocomial respiratory tract infections, complicatedintra-abdominal infections and enterococcal endocarditis 80, 86-93. Synergismpresumably arises as the result of enhanced intracellular uptake ofaminoglycosides caused by the increased permeability of bacteria afterincubation with cell wall synthesis inhibitors such as ?-lactams andglycopeptides 91, 94, 95. Resistance rarely develops during the course oftreatment 96, 97. Gentamicin is the aminoglycoside most often used, becauseof its low cost and reliable activity against Gram negative aerobes 98.

Themajor limitations of aminoglycosides is a relatively low therapeutic index withboth nephrotoxicity and ototoxicity, and that they are not absorbed orally dueto their cationic nature and thus must be given parentally by either anintravenous or intramuscular route 96, 98.  Cell wall active antimicrobials such as ?-lactamsand glycopeptides act by inhibiting the synthesis of the peptidoglycan layer ofbacterial cell walls 99, 100. Penicillins are considered bacteriostaticagainst enterococci, and are the most widely used antimicrobials in the world101. Glycopeptides only work on Gram positive bacteria and is consideredbacteriostatic against enterococci 3, 102. In the last decade severalantimicrobials with effect on enterococci have emerged. They all exhibit lessthan 100% clinical and microbiological success, usually around 70% 80, 103.To improve their efficacy and reduce the development of resistance, it ispreferable to employ them as part of a combination regimen 80, 103. Linezolidinhibits protein synthesis and is active against all clinically important Grampositive bacteria, although it only displays a bacteriostatic effect 104,105.

Daptomycin interferes with the cytoplasmic membrane causingdepolarization and cessation of protein-, DNA and RNA-synthesis 106, 107. Ithas concentration-dependent bactericidal activity against enterococci 108,109. Quinupristin-dalfopristin (Q/D) is a streptogramin antibiotic that isonly active against E. faecium. It inhibits protein synthesis and is consideredbacteriostatic against enterococci 110. Tigecyclin is a broad-spectrumantibiotic that inhibits the protein synthesis. A recent review showed that itwas more effective against enterococci than other Gram positive bacteria, butinfections included were mostly skin and soft tissue infections and intra-abdominalinfections 111, 112.

   ANTIMICROBIAL RESISTANCE IN ENTEROCOCCI The discovery of antibiotics is considered one ofthe most significant health related events of modern times and antibiotictherapy is one of the cornerstones in modern medicine. Use and misuse ofantimicrobials in human medicine and animal husbandry over the past 70 yearshave caused an unremitting selection pressure that has given rise toinnumerable microorganisms resistant to these medicines. The use ofantimicrobials are positively correlated to the emergence of resistant bacteria113, 114.

Several bacteria in the hospital setting in many countriesworldwide are now multiresistant 10, 14, leaving few treatment options.Hence, the development of antimicrobial resistance by bacteria constitutes amajor threat to human health (WHO)     INTRINSICRESISTANCEIntrinsic resistance is a species characteristic,and thus present in all members of the species. Enterococci are resistant to most ?-lactam antibiotics due to apenicillin-binding protein (PBP) that has a low affinity for beta-lactam agents115, 116.  For ampicillin,ureidopenicillins, penicillin and imipenem the resistance is only low level. E.faecium  generally display higher MICsthan E.

faecalis 5. Enterococci display low level resistance toaminoglycosides (se later) and lincosamides 5. E. faecalis also possesses anefflux pump conferring resistance to lincosamides and dalfopristin 117. Inaddition, many wild-type enterococci possess endogenous efflux pumps thatexcrete chloramphenicol  making them low levelresistant 118. Most enterococci are susceptible to co-trimoxazole in vitro,but this combination does not work in vivo, because enterococci are able toincorporate exogenous folic acid which enables them to bypass the inhibition offolate synthesis caused by cotrimoxazole 5.      ACQUIREDRESISTANCEA diversity of antimicrobial resistance genes havebeen demonstrated in the human gut microflora 119.

As inhabitants of thehuman intestinal flora, enterococci are in a position to acquire resistancegenes from this  community, thus makingthem notoriously difficult to treat and enabling them to transfer resistancegenes to even more pathogenic bacteria, such as vanA to S. aureus 120, 121.          COMMONPATHOGENIC SPECIES E. FAECIUM  In the lasttwo decades, E. faecium have evolved as a common nosocomial pathogen,increasing the total burden of enterococcal infections and partially replacingE.

faecalis as a cause of HAI 11. In the beginning of the millennium, genotypicpopulation studies 122, 123 showed distinct genetic lineages spreading in thehospital, suggesting the existence of a specific subpopulation of E. faeciumassociated with hospital acquired infections, different from the community andanimal population. Ampicillin resistance and esp (enterococcal surface protein-a putative virulence gene) were the early markers associated with thissubpopulation 146, 148. Later a pathogenicity island (PAI) containing esp124, IS16 29, 125 and quinolone resistance was also linked to these strains126, 127. In addition, putative virulence genes 128, 129, and severalsurface proteins are enriched in this hospital associated subpopulation 130,131. A large genotypic study of population structure, typing over 400 isolatesby Multi locus sequence typing (MLST) and analyzing it with eBURST, confirmedthe existence of a subpopulation of E. faecium representing clinical andhospital outbreak strains 132.

It demonstrated genetic clustering of hospitalassociated strains, named clonal complex 17 (CC17) that was strongly associatedwith ampicillin resistance and the esp containing PAI. ST17 was presumed to bethe founder of this clonal complex.  Amicroarraybased comparative genomic hybridization of mixed whole genomes,hybridized against 97 isolates also supported the presence of a distinctphylogenetic group of hospital associated strains 29. Many publicationsworldwide have acknowledged CC17 as by far the most prevalent geneticsubcluster causing hospital acquired infections 51, 128, 133-135. The sevenmajor hospital associated STs (ST16, ST17, ST18, ST78, ST192, ST202 and ST203)accounts for 56% of the hospital associated isolates 136.

Later it has beenreported that eBURST based clustering of MLST data to determine evolutionarydecent is inaccurate in species with high levels of recombination such as E.faecium 137. By using other approaches such as ClonalFrame 138 basedphylogenetic trees, constructed from the concatenation of the seven MLSThousekeeping genes 139, or a Bayesian modeling approach using BAPS software 161,165, it has been showed that the CC17 subpopulation has not recently evolvedfrom a single common ancestor; the hospital associated subpopulation  is not clonal (ST17 is not the founder), butrather polyclonal. This polyclonal subpopulation constitutes several lineagesthat seem to have co-evolved into the clade now commonly known as hospital associated E. faecium. A recent study thatinferred phylogeny from 21 publically available E.

faecium genomes by aligning100 orthologs, showed a distinct separation of community-associated andhospital associated strains. They estimated the two lineages to have divergedover 300 000 years ago 33.  Thehospital adapted subpopulation of E. faecium seems to have exploited a novelecological niche- the hospital setting. They seem to be less fit when livingoutside 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 oftenstarts with adaptive changes 141.

Exactly which traits have given thesestrains the upper hand in the hospital setting is not known, but severalproperties have been suggested. Ampicillin resistance is one of the markersstrongest associated with this subpopulation, thus it is suggested that theacquisition of ampicillin resistance was one of the vital traits enabling thestrains to enter the hospitals and evolve into successful nosocomial pathogens125.  This type of adaptive change maygive rise to an amplifying selective process where isolates with the adaptivechange (e.g. ampicillin resistance), more easily can acquire additionaladaptive changes (e.

g. changes in metabolism and other cellular processes)improving their relative fitness 142, 144. The flexibility of the E. faeciumgenome is believed to significantly contribute to the hospital adaptation.Mobile genetic elements (MGE), particularly IS elements are believed toincrease the genome plasticity and facilitate adaptive changes, thus enhancingthe genetic variability in the hospital adapted strains 29, 32. In the lastyears it has become apparent that megaplasmids are abundant among E. faecium,suggesting they play a role in the adaptation of E. faecium to particular hosts49,.

Considering that megaplasmids had not been recognized among enterococcibefore the 1990s 145, and have been shown to play a role in bothcolonization, virulence and resistance in hospital associated E. faecium (selater) 145 they may have played an important role in the recent success ofthese strains.   E.FAECALISThe available data indicates that E. faecalis has anepidemic population structure dominated by a limited number of genetic lineageswith an overrepresentation of clonal complexes CC2, CC9, CC10, CC16, CC21,CC30,CC40 and CC87 13, 176-178. CC2, CC9 and CC87 are considered high riskCCs, as they are enriched in multidrug resistant isolates causing infections inhospitalized patients 13, 168, 176.

CC2 is a globally dispersed hospitalassociated lineage highly capable of causing infections 13, 178-180. Solheimet 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 tobe located within mobile elements such as phage03, a putative integrativeconjugative element and a vanB associated genomic island 142.

CC87 isparticularly dominating in Poland 181, but are also found in other Europeancountries as well as in the US 13. CC9 is spread globally, but high rateshave especially been reported in Spain 17.  The seven most prevalent STs among clinical andoutbreak-associated E.

faecalis (ST6, ST9, ST16, ST21, ST28 ST40 and ST87),account for only 37% of the hospital associated isolates 136. In contrastthis 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 inthe community, including farm animals and food products isolates 13,, indicativeof a reduced host specificity. It has been shown that near 60% of patientsdiagnosed with Vancomycin resistant (VR) E. faecalis bacteraemia in an UShospital, where infected prior to hospitalisation, and that bacteraemia causedby VR E. faecalis was significantly more likely to be present on admission thanbacteraemia caused by VR E. faecium . A recent study showed that CC21, CC16 andCC40 showed better in vitro fitness than those linked to nosocomial infections(CC2, CC9, CC87)  This indicates that hospitalassociated CCs have acquired genetic elements, encoding specific traits(antibiotic resistance, virulence genes) making them successful in the hospitalenvironment, but less fit in the environment. The most recent study on E.

faecalis population structure of human isolates 13 showed that CC2 and CC87were found exclusively in hospitals. It also showed that the six most commonlydetected CCs (CC2, CC16, CC21, CC30, CC40 and CC87) accounted for 57% of thehospital isolates. Comparison of gene tree topologies of individual MLST genesindicates that recombination rates in E. faecalis are even higher than in E.faecium. Hence, recombination seems to be the driving force in diversificationand evolution of this species 143.

Thus it is may be more accurate toconsider CCs rather than STs as genetic lineages in E. faecalis.