Revisão Enterococcus

7/29/2019 Reviso Enterococcus

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Enterococci have been known for more than a centuryfor their role as a common cause of enocaritis 1, a is-ease that is fatal without effective antimicrobial therapy.The enterococci are Gram-positive, facultatively anaer-obic oval cocci that form chains of various lengths; theyare stury an versatile, with a particular ability to sur-

vive uner harsh conitions (incluing high salt concen-trations) an at a wie range of temperatures (from 10 Cto >45 C). The first escription of an enterococcal infec-tion namely, infective enocaritis ates from1899 (REF. 1), an enterococci were subsequently shownto cause a range of infections in the community setting(incluing pelvic infections, neonatal infections anurinary tract infections (UTIs)), as well as infective eno-

caritis. However, in spite of their pathogenic potential,enterococci generally isplay low levels of virulence,as evience by their presence as natural colonizers ofthe gastrointestinal (GI) tract in most humans an ani-mals an by the fact that they have been use safely forecaes as probiotics in humans an farm animals.

Both microbial an host factors can contribute to theconversion of a secon-rate pathogen into a first-rateclinical problem. For the enterococci, such factors appearto inclue their inherent ability to resist anti microbialagents (for example, clinamycin, cephalosporins anaminoglycosies), their capacity to acquire an issemi-nate eterminants of antibiotic resistance (for example,

vancomycin resistance gene clusters) an their malleablegenomes, which may contribute to their aaptation toharsh environments (incluing hospitals) an increasethe ability of certain lineages to colonize the GI tractan/or isseminate outsie the bowel. Moreover, theincreasing number of patients who are hospitalize incritical care units an are immunosuppresse, mechan-ically compromise (by catheters, for example) anreceiving multiple antimicrobial agents favours the abil-ity of multirug-resistant organisms such as enterococcito cause isease. In this Review, we iscuss the factorsthat may have contribute to the rise of enterococci asnosocomial pathogens, with an emphasis on the epiemi-ology an pathogenesis of infections by these species, an

on mechanisms of resistance to the most relevant anti-enterococcal agents use in clinical practice. For a moreetaile iscussion of the clinical an therapeutic aspectsof enterococcal infections, the reaer is irecte to otherrecent reviews2,3.

The epidemiology of enterococcal infections

In hospitals in the Unite States, enterococci are the sec-on most common organisms recovere from catheter-associate infections of the bloostream an uri-nary tract, an from skin an soft-tissue infections2,4.Hospital-associate enterococcal infections in theUnite States have emerge in two istinct waves. The

1Department of Internal

Medicine, Division of

Infectious Diseases, Center

for the Study of Emerging and

Re-emerging Pathogens,

Houston, Texas 77030, USA.2Laboratory for Antimicrobial

Research, University of Texas

Medical School at Houston.3Molecular Genetics and

Antimicrobial Resistance Unit,

Universidad El Bosque,

Carrera 7B Bis # 132-11,

Edificio Rectoria, Bogot,Colombia.4Laboratory of Enterococcal

Research, University of Texas

Medical School at Houston.5Department of Microbiology

and Molecular Genetics,

University of Texas Medical

School at Houston,

6431 Fannin Street,

Room 2.112 MSB,

Houston, Texas 77030, USA.

e-mails:

[email protected];

[email protected]

doi:10.1038/nrmicro2761

The rise of the Enterococcus:beyond vancomycin resistanceCesar A. Arias1,2,3 and Barbara E. Murray1,4,5

Abstract | The genus Enterococcus includes some of the most important nosocomial

multidrug-resistant organisms, and these pathogens usually affect patients who are

debilitated by other, concurrent illnesses and undergoing prolonged hospitalization. This

Review discusses the factors involved in the changing epidemiology of enterococcal

infections, with an emphasis on Enterococcus faecium as an emergent and challengingnosocomial problem. The effects of antibiotics on the gut microbiota and on colonization

with vancomycin-resistant enterococci are highlighted, including how enterococci benefit

from the antibiotic-mediated eradication of Gram-negative members of the gut microbiota.

Analyses of enterococcal genomes indicate that there are certain genetic lineages, including

an E. faecium clade of ancient origin, with the ability to succeed in the hospital environment,

and the possible virulence determinants that are found in these genetic lineages are

discussed. Finally, we review the most important mechanisms of resistance to the antibiotics

that are used to treat vancomycin-resistant enterococci.

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Perineal skin

Environmental contamination

Catheter

Heart valves

Liver

Bloodstream

Gut

ProbioticsMicroorganisms such as

bacteria and yeasts that,

when ingested, are thought to

provide a beneficial effect

to the host.

Third-generationcephalosporins-lactam compounds with

broad-spectrum activity

against Gram-positive cocci

(except enterococci) and many

Gram-negative bacteria. These

antibiotics include ceftazidime,

ceftriaxone, cefotaxime and

cefpodoxime.

LipopolysaccharideAn important component of

the outer membrane of

Gram-negative organisms,

composed of a membrane lipid

(lipid A) and a polysaccharide

chain.

FlagellinA protein that forms the

flagellum of Gram-negative

bacteria.

Toll-like receptorsReceptors that are found on

and in eukaryotic cells and

recognize molecular patterns

which are shared by bacterial

pathogens.

LectinA protein that binds to

carbohydrate moieties.

first wave began in the late 1970s an was associatewith the introuction ofthird-generation cephalosporins5;uring this era, Enterococcus faecalis accounte for9095 % of clinical enterococcal isolates. We are now inthe mist of the secon wave, cause byEnterococcusfaecium, which is much more frequently resistant tovancomycin an ampicillin than E. faecalis. This wavehas increase steaily since the early 1990s in hospitalsin the Unite States, but is now also present in otherparts of the worl4,6 an is associate with the increaseuse of vancomycin an broa-spectrum antibiotics.Inee, the number of infections with vancomycin-resistant enterococci (VRE) in US hospitals increasefrom 9,820 in 2000 to 21,352 in 2006 (REF. 7); further-more, E. faecium is now almost as common a cause ofnosocomial infections as E. faecalis4. This change inspecies is of paramount clinical importance, as E.fae-cium is by far the more ifficult of the two species totreat. For example, in the Unite States, the percentageofE. faecium isolates that were resistant to vancomycin

rose from 0 % before the mi 1980s to more than 80 % by2007 (REF. 2); by contrast, only ~5 % ofE. faecalis isolatesare vancomycin resistant4.

Multirug-resistant enterococci are currently less of aproblem outsie the Unite States. The initial reports ofVRE in Europe in the late 1980s were of organisms thatwere mostly colonizers of the GI tract of animals anhumans in the community. Inee, a strong correlationbetween the use of avoparcin, a glycopeptie antibioticthat was use in animal fee, an the emergence of VREin Europe le to the ban of this compoun from animalhusbanry in 1996 (REF. 8). Although a ecrease in theprevalence of VRE in animals in Europe was initially

observe after the avoparcin ban, subsequent surveil-lance has reveale an increase in nosocomial ampicillin-an/or vancomycin-resistant enterococcal infectionsover the past ecae. For example, in the Netherlans,the average number of invasive ampicillin-resistantenterococcal infections per hospital increase from~10 infections in 1999 to ~50 in 2005 (REF. 9). By 2007,

vancomycin resistance among clinical enterococcal iso-lates from Europe varie from >30 % in countries suchas Greece an Irelan to less than 1 % in Scaninaviancountries8, although a recent alarming report fromSween ocumente an approximately fourfol increasein infections by VRE in the perio 20072009 com-pare with 20002006 (REF. 10). In Latin America, amulticentric prospective stuy in four countries founthat the majority (~78 %) of enterococcal infections arestill cause by ampicillin- an vancomycin-susceptibleE. faecalis, although the remaining ~22 % are cause bygenetic lineages of multirug-resistant E. faecium thatare similar to those observe in the Unite States 11. InAsia, the number of infections by vancomycin-resistant

E. faecium is still low, although outbreaks have beenocumente12.

Colonization of the GI tract. Enterococci are commoninhabitants of the GI tract of humans an other animalsas well as that of insects an nematoes. Although thesespecies normally constitute a small proportion of thegut microbiota13, an important first step towars noso-comial enterococcal infection seems to be increaseensity of colonization of the GI tract (FIG. 1). The expo-sure of hospitalize patients to antibiotics (for example,cephalosporins an some penicillins that is, piperacillintazobactam with activity against Gram-negative bac-teria an Gram-positive species excluing E. faecium)results in substantial changes in the gut microbiotathat facilitate colonization of the GI tract by VRE14,15.Pioneering work using mouse moels of intestinalcolonization16 has provie consierable insights into themechanisms by which VRE gain the upper han inthe gut after antibiotic exposure. These stuies founthat lipopolysaccharide an flagellin of Gram-negativebacteria, incluing anaerobes, stimulate (via inter-actions with Toll-like receptors)16,17 the prouction ofREGIII by Paneth cells; REGIII is a C-type lectin withanti microbial activity against Gram-positive bacteria,incluing VRE16. Depletion of the Gram-negative micro-biota by antibiotics ecreases the prouction of REGIII

an thus facilitates overgrowth of VRE16,17 in the GI tract.This highlights the role of the intestinal microbiota inmoulating colonization by multirug-resistant organ-isms (FIG. 2). Inee, the use of the antibiotics metro-niazole, neomycin an vancomycin in mice allowsVRE to become the preominant intestinal species anto remain ominant for up to 2 months after the anti-biotic regimen is iscontinue15. Extening this work tohumans, it was shown that invasion of the bloostreamof hospitalize patients by VRE is precee by VREbecoming the preominant species in the GI tract ofthese patients15. In aition to the intestinal changes, the

vancomycin resistance an the high minimal inhibitory

Figure 1 | The crucial role of the gastrointestinal tract

in enterococcal infection and spread. Enterococci from

the gastrointestinal tract can access the bloodstream by

moving across the intestinal lining and passing through the

liver. When in the bloodstream, these organisms can reach

the heart and then potentially cause infective endocarditis.Faecal contamination of the environment (which can then

be a source for colonization of other patients) and of the

patients skin (the main source of infections of the urinary

tract and of intravenous catheters) frequently occurs.

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TLR4?

TLR5

a

No antibiotics

Intestinal lumen

b c

Paneth cellIL-22Epithelialcell

REGIII+ REGIII

Antibiotics

Gram-positive organisms

REGIII

REGIII

Gram-negative organisms

Clonal complexesGroups of bacterial isolates,

as derived from multilocus

sequence typing (MLST)

analysis. A clonal complex

usually includes isolates that

differ from one another at only

one of the seven loci analysed

by MLST.

CladeA group of bacterial isolates

that are genetically related

and probably share a common

ancestor.

concentrations of piperacillin an cephalosporins is-playe by ampicillin-resistant E. faecium allow theseorganisms to survive in the gut of patients who are beingtreate with these antibiotics.

Nosocomial transmission of VRE. Enterococci are toughbugs that can survive for long perios on environmentalsurfaces, incluing meical equipment, be rails anoorknobs18(FIG. 3). They are tolerant to heat, chlorinean some alcohol preparations18, which may help explainwhy these organisms are wiely isseminate in the hos-pital setting. There are several risk factors for evelopinga nosocomial VRE infection: close physical proximity topatients infecte or colonize with VRE; a long perioof hospitalization; multiple courses of antimicrobials;hospitalization in long-term facilities, surgical units orintensive-care units; soli organ an bone marrow trans-plantation; co-morbiities such as iabetes, renal failureor haemoialysis; an the presence of a urinary catheter19.

An unerstaning of the transmission ynamics ofenterococci in the hospital environment is pivotal forinfection control. Hospitalize patients often receiveantimicrobials that increase the ensity of VRE in the GItract, in turn facilitating the sprea of these organisms.A mathematical moel20 of transmission rew parallelsbetween VRE transmission an the transmission of a

vector-borne infection such as malaria. In this moel,health care personnel play the part of the mosquito, car-rying VRE on their hans from patients who are VREpositive to those who are VRE negative, an to their sur-rounings, an there is a potential to transmit the patho-gens with each contact. Transmission can be amplifie

epening on how many patients have contact with thevector (REF. 20) an correlates with the ensity of VREin patient stools. This concept highlights the importanceof curtailing the chain of transmission through activesurveillance an contact precautions for infecte ancolonize iniviuals, through implementation of stricthan hygiene practices for health care workers, through

juicious use of antimicrobials (via programmes of anti-biotic stewarship) an through aggressive environmentalcleaning methos19,21.

Population genetics of enterococci. Early stuies on themolecular epiemiology ofE. faecalis22 an E. faecium23using pulse-fiel gel electrophoresis (PFGE) showe thesprea of single clones to ifferent US states. Multilocussequence typing (MLST), which is base on allelic if-ferences in seven housekeeping genes an is preferrefor analyses of ancestral relationships, helpe establishthat enterococcal isolates recovere from hospitalize

patients often cluster in specific groups (esignate clonalcomplexes) that are ifferent from those of commensalisolates or isolates recovere from animals. Inee, themajority of hospital-erive isolates ofE. faecalis clusterin two clonal complexes, CC2 an CC9 (REFS 24,25), anthe increase frequency of isolation ofE. faecium worl-wie is ue to the presence of a polyclonal subpopulation(particularly MLST sequence type 17 (ST17), ST18, ST78an ST192, which were previously esignate clonal com-plex CC17)6,26; recent comparisons of available genomesequences support the concept of a hospital-associateclade that is genetically istinct from most commensalisolates from animals an humans6,2729.

Figure 2 | The effects of antibiotic administration on the gastrointestinal microbiota and the emergence of

vancomycin-resistant enterococci. a | In the absence of antibiotics, mouse intestinal epithelial cells and Paneth

cells produce the C-type lectin REGIII, which has antimicrobial activity against Gram-positive bacteria (purple). The

production of REGIII is triggered by the presence of Gram-negative bacteria (pink); their MAMPs (microorganism-associatedmolecular patterns), such as the outer-membrane lipopolysaccharide (in the intestinal lumen) and flagellin (in subepithelialtissues), are recognized by pattern recognition receptors such as Toll-like receptor 4 (TLR4) and TLR5, respectively.

b | Antibiotic administration leads to a reduction in the Gram-negative bacteria, which decreases REGIII production byintestinal epithelial cells and Paneth cells. c | Enterococci take advantage of this reduction in REGIII secretion to becomethe dominant members of the gut microbiota. IL-22, interleukin-22. Figure is modified, with permission, from REF. 144

(2010) American Society for Clinical Investigation.

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Contaminated hands ofstaff and visitors

Contaminated equipment

Colonized patient

Faecalcontamination

Oral

acquisition

IV antibiotic

Contaminated door handles,

sinks, toilets, bed rails and

other communal surfaces

Susceptible patient

Pheromone-responsiveplasmidsPlasmids that are known

to respond to a peptide

pheromone produced by a

recipient cell, which initiates

the mating process.

In a recent comparison of 100 core genes from avail-able E. faecium genomes29, it was foun that most (91) ofthese genes split into two groups that iffer by 3.54 %,confirming that there are inee two ancestral claesofE. faecium. The hospital-associate group containsmost of the E. faecium clinical isolates that have beensequence, although it also contains isolates of commu-nity origin; the community-associate group consistsalmost entirely of isolates from the community (fromboth humans an animals). In aition to the ifferencesbetween the genes of the two claes, molecular-clockanalysis estimate that these groups iverge from each

other at least 300,000 years ago. As previously reporte,insertion sequence 16 (IS16)27 an the gene encoing theampicillin-resistant penicillin-bining protein 5 (pbp5R)are consistently foun in the hospital-associate strains,whereas the gene encoing the ampicillin-sensitive PBP5(pbp5S) is present in the community-associate clae30.

One of the main challenges in the analysis of entero-cocci is the enormous plasticity of their genomes. In bothE. faecalis an E. faecium, acquire elements can accountfor up to 25 % of the genome28,3137. Moreover, in a seriesof elegant experiments, it has been shown that conjuga-tion (bacterial mating) between enterococci can proucetransconjugant strains with hybri genomes; that is, large

fragments (up to 800 kb) of chromosomal DNA can betransferre from a onor strain to a recipient strain ancan result in changes in the MLST pattern38. The transferepens on the presence ofpheromone-responsive plasmidsthat can insert into the chromosome, presumably byrecombination between homologous sequences on theplasmi an chromosome38 (reviewe in REFS 33,39).Evience of recombination in nature was obtaine by arecent analysis which note that in some of the analysestrains, all of the core genes are from one clae, but in themajority of analyse strains, between one an eight geneshave been replace with the corresponing gene from the

other clae29. In one strain, which was consiere in thisanalysis to be a true hybri strain, 26 of the 92 genes ana-lyse belong to the community-associate group, whereasthe rest belong to the hospital-associate clae29.

Another important component in the evolution ofmultirug-resistant strains of hospital-associate entero-cocci may be their lack of CRISPR (clustere regularlyinterspace short palinromic repeats) elements, whichprovie bacteria with a efence system against incomingDNA. Inee, the presence of antibiotic resistanceeterminants is inversely correlate with the presenceof CRISPRs in E. faecalis, an most isolates fromhospital-associate clonal complexes lack CRISPRs40.

Figure 3 | Major routes of nosocomial transmission of vancomycin-resistant enterococci. The main risk factors

for colonization and subsequent nosocomial infection with vancomycin-resistant enterococci (VRE) include close

physical proximity to patients who are infected or colonized with VRE (or to the rooms of these patients); a long period

of hospitalization; hospitalization in long-term facilities, surgical units or intensive-care units; the presence of a urinary

catheter; and the administration of multiple courses of antibiotics. Many antibiotics increase the density of VRE organisms

in the gastrointestinal tract, which, in turn, facilitates the spread of these organisms through faecal contamination of the

hospital environment, including inanimate objects and the hands of health care workers and vistors. Enterococci can

survive for long periods on environmental surfaces, including medical equipment, toilets, bed rails and doorknobs, and

are tolerant to heat, chlorine and some alcohol preparations. IV, intravenous.

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Aggregation substanceproteinsA family of surface-localized

proteins that are encoded

by pheromone-responsive

plasmids. These proteins

mediate binding of donor

bacterial cells to recipients

cells.

LPXTG-like motifsSpecific amino acid sequences

(X refers to any amino acid)

that are found in surface

proteins and are necessary

for the specific attachment of

the protein to the cell wall

peptidoglycan.

Immunoglobulin-like foldsStructural domains of

immunoglobulins, consisting

of two sheets of antiparallel

strands that form a

sandwich-like structure. Thesefolds are shared by some

bacterial surface proteins.

PseudogeneA gene that has lost its ability

to be expressed or to be

functional.

PiliHair-like projections that are

present on the bacterial

surface.

Pilin subunitsProteins that form the pilus.

Enterococcal pathogenesis

The rise of enterococci as nosocomial pathogens wasinitially thought to have resulte entirely from the selec-tive avantage provie by their resistance to antibiotics.However, it is likely that other virulence eterminantsare also involve in the success of these microorganismsin the hospital setting. Research into the mechanismsby which enterococci cause isease has yiele impor-tant insights into their biology. Unlike streptococci anstaphylococci, most enterococci o not prouce a set ofpotent pro-inflammatory toxins, but they are equippewith many genes encoing ahesion proteins that maymeiate aherence to host tissues, consistent with theirpathogenic role in infective enocaritis. An extenereview of all the potential pathogenic factors ientifiein enterococci is beyon the scope of this Review, anhere we concentrate on those that have been shown tohave an important role in experimental mammalianmoels (TABLE 1).

Secreted factors. Several proteins that are secrete

into the extracellular meium have been implicate inenterococcal virulence. Haemolysincytolysin (Cyl) isa toxin prouce by ~30 % ofE. faecalis strains anis encoe on pheromone-responsive plasmis orpathogenicity islans. Cyl is secrete extracellularly astwo structural subunits (CylL-L an CylL-S) an thenproteolytically activate41,42. Cyl can lyse re bloo cellsfrom humans, horses an rabbits, but not sheep or cows,an can also lyse some human white bloo cells43. E. faec-alis strains expressing cylare more virulent in variousanimal moels than isogenic strains without cyl43.

Other important secrete factors inclue theproteases gelatinase (GelE) an extracellular serineproteinase (SprE). GelE seems to meiate virulencethrough effects such as egraation of host tissuesan moulation of the host immune response44. It hasan important role in clearing misfole proteins45 anparticipates in the activation of autolysin, a peptio-glycan-egraing enzyme46, which leas to the releaseof extracellular DNA an the formation of a biofilm. Thegenes encoing these proteases in E. faecalis are regulateby the Fsr quorum sensing system47, which is homolo-gous to the Agr system of staphylococci (involve inregulating the expression of several virulence factorsin Staphylococcus aureus) an has been shown to affectthe pathogenesis of some E. faecalis infections48,49.Mutants lacking GelE show a marke ecrease in biofilm

formation50, a ecrease in translocation across T84 intes-tinal cells51, attenuate virulence in peritonitis, enocar-itis, enophthalmitis an C. elegans moels5256, anreuce aherence to ental roots57. It shoul be notethat Cyl an GelE are seen equally in isolates recov-ere from clinical infections an in those from stoolsof healthy iniviuals, illustrating that various putativeenterococcal virulence eterminants can be also founin strains colonizing the GI tract of healthy iniviuals58.

Cell surface determinants. One group ofE. faecalis sur-face proteins that has been extensively characterize isthe family ofaggregation substanceproteins (AS proteins).

These proteins are encoe by pheromone-responsiveplasmis that often also harbour antibiotic resistancegenes. AS proteins cause clumping of E. faecalis cellsan meiate high-frequency transfer of plasmi DNA inliqui meia. These proteins increase E. faecalis bin-ing to culture renal epithelial cells, survival withinpolymorphonuclear neutrophils an internalizationby intestinal cells59, an they affect the pathogenesis ofexperimental enocaritis (presumably by favouring theformation of large bacterial aggregates on the cariac

valve)60,61. The enterococcal surface proteins Esp anEsp

fm, encoe by genes that seem to have been acquire

within a pathogencity islan, are commonly foun inclinical isolates, are anchore to the cell wall, affect bio-film formation62 an have a role in experimental UTIan/or enocaritis moels6365.

Enterococcal MSCRAMMs (microbial surface com-ponents recognizing ahesive matrix molecules) areconsiere to be important elements in the early stagesof infection, as they may bin components of the hostextracellular matrix. Although many putative ahesins

have been escribe in both E. faecalis an E. faecium,the most extensively stuie are the collagen ahesinsAce in E. faecalis an Acm in E. faecium. Both are cellwall-anchore ahesins that contain LPXTG-like motifs (aso AS proteins an Esp), have immunoglobulin-like folds66an have consierable similarity with the S. aureus col-lagen ahesin (Cna) (there is a 62 % overall similaritybetween Acm an Cna)67. They have both been shown toaffect the pathogenesis of enterococcal infections in vivo.E. faecalis Ace is a collagen an laminin ahesin thatis conitionally expresse after growth in collagen orserum68 an bins collagen in a specific manner referreto as the collagen hug (REF. 69), in which Ace appears toembrace the collagen molecule after initial ocking.An ace eletion mutant is substantially attenuate inmoels of enocaritis70 an UTIs71,72 compare with theparental strain, an antiboies specific for the collagen-bining omain of Ace protect rats from enocaritis70.E. faecium Acm bins collagen, an an acm eletionmutant is attenuate in experimental enocaritis73.Community-associate strains (isolates from animalan human stools), unlike hospital-associate strains,often have an acmpseudogene an rarely express Acmor ahere to collagen. Thus, Acm may have a role in theincrease ability of members of the hospital-associateE. faecium clae to cause isease73.

Another enterococcal surface protein, ElrA (entero-

coccal leucine-rich-repeat-containing protein), is a cellwall-associate protein from the WxL family. An E. faec-aliselrA eletion mutant has attenuate virulence in amouse peritonitis moel an cannot infect macrophages(this is associate with a ecrease in the interleukin-6response)74.

Some enterococcal proteins with LPXTG-like motifsan immunoglobulin-like fols are tethere into pili,which are filamentous structures protruing from thecell surface75,76. Similar pili that are present in otherGram-positive organisms have an important role ininfection an colonization of host tissues77, an typi-cally contain two or three pilin subunits that are encoe

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SortaseAn enzyme for which the main

function is to attach surface

proteins to the cell wall

peptidoglycan or to other

proteins after recognition of

an LPXTG motif.

in loci which also harbour one or more pilus-specificsortase genes75. Following assembly of the pilin subunitsby the pilus-specific sortase, a housekeeping sortase thenattaches the pilus to the cell wall. Ebp (enocaritis anbiofilm-associate pili) pili are ubiquitous in E. faeca-lis; their expression is represse by the Fsr system78 anactivate by RnjB, an RNase of the J2 family79. Ebp pili

are important for biofilm formation an for the patho-genesis of experimental enocaritis an UTIs80. Onlyone other pilus locus, esignate biofilm enhancer inEnterococcus (bee), has been foun in E. faecalis, but thislocus is rare81, whereas E. faecium often harbours four ormore putative pilus loci82, two of which have been con-firme to prouce pili76. One of these loci has homology

Table 1 | Enterococcal determinants that have been shown to cause virulence phenotypes in vivo

Determinant Possible function and role

Secreted factors

Cyl Increases virulence ofE. faecalis after intraperitoneal inoculation in mice43

Role in experimental endophthalmitis145

Lysis of red blood cells43, retinal cells, polymorphonuclear neutrophils andmacrophages

GelE Role in experimental endocarditis55,56, peritonitis52 and endophthalmitis54

Role in clearing misfolded proteins from the bacterial cell61

Affects translocation across intestinal epithelial cells (in vitro)51

Activates autolysin and biofilm formation46

Affects adherence to dental roots57

Regulated by the Fsr quorum sensing system47

Cell surface determinants and their formation

AS proteins Promote transfer and survival in neutrophils for some plasmids and affectinternalization ofE. faecalis by epithelial cells61

Role in E. faecalis experimental endocarditis60

Esp (E. faecalis) and Espfm

(E. faecium)Affect biofilm formation62

Role in experimental UTIs and/or endocarditis6365

Ace (E. faecalis) and Acm(E. faecium)

Role in experimental endocarditis and UTIs7072

Mediate adherence to collagen (Acm), and to collagen and laminin (Ace)68

ElrA Role in experimental peritonitis (E. faecalis)74

Affects the ability to infect macrophages74

Ebp (E. faecalis) and Ebpfm

(E. faecium) proteins

Form pili75,83 and play a part in experimental endocarditis and/or UTIs75,80

Affect biofilm formation75,83

Mediate adherence to platelets, fibrinogen and collagen (E. faecalis)146

SrtA Role in catheter-associated UTIs147

Important for biofilm formation147

epa cluster Affect biofilm formation88 and translocation across intestinal epithelial cells89

Role in experimental peritonitis and UTIs89,92

Affect bacterial cell susceptibility to killing by polymorphonuclear neutrophils90

DGlcDAG glycolipid Role in experimental bacteraemia94

Affects biofilm formation and adherence to intestinal epithelial cells94

Other factors

Megaplasmids (E. faecium) Affect colonization of the mouse gastrointestinal tract98

Role in experimental peritonitis97

Gls24 (E. faecalis), and Gls20and Gls33 (E. faecium)

Role in experimental peritonitis101 and, for Gls24, also in endocarditis100

Peroxidases such as Tpx(E. faecalis)

Role in experimental peritonitis103

Protection within the phagocyte environment103

MsrAB (E. faecalis) Role in experimental UTIs148

Affect survival in peritoneal macrophages148

PerA (E. faecalis) Role in experimental peritonitis149

Role in biofilm formation, and affects survival within macrophages in vitro

Bop Contributes to prolonged mouse bacteraemia and biofilm formation150

SigV Role in mouse UTIs and experimental mouse bacteraemia151

Ace, adhesin of collagen from E. faecalis; Acm, adhesin of collagen from E. faecium; AS, aggregation substance; Bop,biofilm onplastic surfaces; Cyl, haemolysincytolysin; DGlcDAG, -diglycosyl diacylglycerol; Ebp, endocarditis- and biofilm-associated pili;Ebp

fm, E. faecium Ebp; E. faecalis, Enterococcus faecalis; E. faecium, Enterococcus faecium; ElrA, enterococcal leucine-rich-repeat-

containing protein; epa, enterococcal polysaccharide antigen; Esp, enterococcal surface protein; Espfm

, E. faecium Esp; GelE,gelatinase; MsrAB, methionine sulphoxide reductase AB; PerA, pathogenicity island-encoded regulator; SigV, extracytoplasmicfunction -factor; SrtA, sortase A; Tpx, thiol peroxidase; UTI, urinary tract infection.

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OpsonicReferring to the ability of a

moiety to increase phagocytosis

of an invading pathogen.

Lipoteichoic acidAn important structure of thecell wall of Gram-positive

bacteria. It is composed of cell

membrane-anchored teichoic

acids (chains of ribitol

phosphate).

-lactam antibioticsNaturally produced

antimicrobials encompassing

several classes, including

penicillins, cephalosporins,

cephamycins, monobactams

and carbapenems, all of which

contain a -lactam ring.

to E. faecalis Ebp an also affects biofilm formation anthe pathogenesis of mouse UTIs83.

Polysaccharies are important components ofthe Gram-positive cell surface an have a crucial rolein pathogenesis, meiating evasion of phagocytosisby polymorphonuclear neutrophilsan stimulatingcytokine prouction84. Some E. faecalis strains, especiallythose belonging to hospital-associate clonal clusters,have a capsular polysaccharie locus (cps) containing89 genes. Isolates that are esignate polysaccharietype A an type B lack cps genes, an it was shown thatthe opsonic antiboies use to classify type A isolatesactually recognize lipoteichoic acid (LTA)85. Type C an Dpolysaccharies mask LTA an confer resistance tocomplement-meiate opsonophagocytosis86,87.

Enterococcal polysaccharie antigen (Epa), a 37 kDacell wall antigen, is recognize by sera from most patientswith serious E. faecalis infections88. Epa is a rhamnose-containing polysaccharie, biosynthesis of which isencoe by the epa locus (containing 16 genes), an itis postulate to be cell wall associate89. Disruption of

the epa cluster ecreases biofilm formation an translo-cation across an enterocyte monolayer90, increases sus-ceptibility to polymorphonuclear neutrophil-meiatekilling91, an causes attenuation in mouse peritonitis anUTI moels92.

Enterococcal cell membrane glycolipis an LTA93also appear to be important in pathogenesis. An E. faeca-lis mutant that is eficient in the glycolipi -iglycosyliacylglycerol (DGlcDAG) shows reuce aherenceto enterocytes an reuce biofilm formation an wascleare faster from the bloostream of mice than wiltype E. faecalis94.

Other influences on virulence. Non-pheromone-responsive, large transferable plasmis, 150250 kbin size, are common among E. faecium clinical iso-lates11,95,96 an have a role in virulence97. Transfer of oneof these megaplasmis from a clinical strain (E. fae-cium str. TX16(DO)) to a commensal E. faecium strainincreases the virulence of the commensal in experi-mental mouse peritonitis97. Similarly, transfer of amegaplasmi from a ifferent E. faecium clinical strainincreases the ability of a laboratory strain (E. faeciumstr. D344SRF) to colonize the GI tract of mice98. Theprecise role of specific genes carrie by these plasmis in

virulence or colonization is unclear, but recent eviencesuggests that one gene, hyl

Efm(encoing a family 84

glycosyltransferase), which is frequently referre toas a virulence factor, is not the main meiator of theincrease virulence that is conferre by the megaplas-mis99, inicating that other genes on these plasmismay offer a competitive avantage.

Some stress response proteins are also importantfor virulence. Gls24 is one such enterococcal protein,meiating resistance ofE. faecalis to bile salts; inter-estingly, Gls24-specific immune serum protects miceagainst a lethal E. faecalis challenge in the peritonitismoel, an agls24 mutant is attenuate in experimentalenocaritis100. Similar proteins in E. faecium have alsobeen shown to be important in mouse peritonitis101. In

aition, E. faecalis prouces three types of peroxiasethat etoxify reactive oxygen species (the meiatorsof bacterial killing by phagocytes): NADH peroxiase(Npr)102,103, Ahp (an alkyl hyroxyperoxie reuctasesystem) an thiol peroxiase (Tpx). Tpx is the mostrelevant peroxiase for protection in the phagocyteenvironment, an a tpx eletion mutant is attenuate inmouse peritonitis103.

Mechanisms of antibiotic resistance

Enterococci are intrinsically resistant to several anti-biotics an also reaily accumulate mutations anexogenous genes that confer aitional resistance.The acquisition of resistance genes often occurs byconjugation using pheromone-responsive plasmis(encoing the AS proteins mentione above) an theirhigh-frequency transfer between E. faecalis isolates104,conjugative plasmis with a broa host range, or conju-gative transposons with the potential to carry multipleantibiotic resistance genes105.

The challenges in the treatment of enterococcal infec-

tions have been apparent since the early ays of peni-cillin use, in the 1940s, when penicillin monotherapywas note to be successful for infective enocaritiscause by streptococci106 an staphylococci, but notenterococci107. As oppose to their effect against moststreptococci, -lactam antibiotics such as penicillins arenot bactericial against many strains of enterococci;moreover, tolerance to penicillins (that is, lack of killingespite growth inhibition) can be rapily elicite in somestrains by intermittent exposure to the antibiotics108.The combination of a penicillin an streptomycin (anaminoglycosie) was empirically foun to be curative inpatients with enterococcal enocaritis an was shownto prouce an synergistic bactericial effect in vitro109(that is, a 3 log10 ecrease overall an a 2 log10 ecrease

versus penicil lin alone 24 hours after exposure); thisantibiotic combination became the stanar treatmentfor enterococcal enocaritis by the 1950s. Many of themoern nosocomial E. faecium isolates are resistant toampicillin an vancomycin, an also have high-levelresistance to aminoglycosies (which eliminates theeffectivity of antibiotic combinations (synergism)).This is one of the most pressing issues currently facingclinicians in hospitals worlwie, as other therapeuticoptions are not reliable, have toxic sie effects or havenot been teste in prospective ranomize clinical tri-als. In this section, we iscuss the major mechanisms of

antibiotic resistance that aversely affect the treatmentof enterococcal infections (FIG. 4).

lactam resistance. Penicillin (or ampicillin) alone orwith an aminoglycosie was the cornerstone for treat-ment of enterococcal infections for more than half acentury. Ampicillin resistance, which is rare in E. faeca-lis, occurs in ~90 % of moern-ay hospital-associateE. faecium isolates.S. aureus commonly uses -lactamaseto overcome penicillin, but this mechanism is rarely seenin enterococci, although there were outbreak strains ofE. faecalis in the 1990s110 that prouce a -lactamaseientical to the staphylococcal enzyme. The presence

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Lipopeptides(daptomycin)

Alteredinteractionwith cellmembrane

GdpD

LiaF

Cls

Aminoglycoside-modifying enzymes

Aminoglycosides(gentamicin andstreptomycin)

Low-level resistance Low uptake

PBP5

-lactams(ampicillin)

Glycopeptides(vancomycin)

Alteredtargetbinding

Reducedbinding

affinity

Reducedbinding

affinity

Alteredtargetbinding

D-Lac

23s rRNA mutationsand/or modifications

Streptogramins(QD)

Inactivation

Cell membrane Peptidoglycan

Modification

Efflux

Oxazolidinones(linezolid)

D-Ala

High-level resistance

Vat

VgbMsrC

of -lactamase is not a therapeutic challenge, as it isinhibite by -lactamase inhibitor combinations (suchas ampicillinsulbactam), but it may pose a iagnosticchallenge, as strains test positive for resistance only athigh inocula. The mechanism responsible for ampicillinresistance in E. faecium is the prouction of PBP5, which

has low affinity for the penicillins (an perhaps is over-prouce)111. The PBP5-encoing gene in ampicillin-resistant strains,pbp5R, is foun in E. faecium isolatesin the hospital-associate clae an iffers by ~5 % frompbp5S, the gene in ampicillin-susceptible strains in thecommunity-associate clae30. In the laboratory,pbp5Rcan transfer between bacterial cells, at times along witha transposon (Tn5382) that carries vancomycin resist-ance genes112, to E. faecium isolates that are more sus-ceptible to ampicillin, probably by a similar mechanismto that use for the transfer of large chromosomal frag-ments between E. faecalis strains38. The crystal structureof PBP5 inicates that specific amino aci ifferences

may be responsible for resistance by interfering withthe architecture of the active site or with -lactam bin-ing113,114. High-level resistance to ampicillin emerge inhospitals in the Unite States in the 1970s to 1980s115,an it was into this ampicillin-resistant E. faecium back-groun that vancomycin resistance first emerge in the

Unite States. Although in Europe vancomycin resist-ance was initially preominant in ampicillin-susceptiblestrains that are now recognize as belonging to the com-munity-associate E. faecium clae, a similar pattern ofemergence of resistance first to ampicillin an thento vancomycin has subsequently been observe inhospital-associate E. faecium from Europe8.

Aminoglycoside resi stance. Although enterococciexhibit intrinsic resistance to low to moerate levels ofaminoglycosies, the uptake of these highly polar mol-ecules116 can be enhance by the aition of cell wall-active agents, incluing penicillins an vancomycin109.

Figure 4 | Main mechanisms of enterococcal antibiotic resistance. Enterococci are intrinsically resistant to several

antibiotics and can acquire mutations and exogenous genes that confer resistance to additional drugs. The main

mechanisms of antibiotic resistance are shown. In Enterococcus faecium, resistance to ampicillin occurs through the

production of penicillin-binding protein 5 (PBP5), which has low affinity for -lactams. Enterococci exhibit intrinsiclow-level resistance to aminoglycosides such as streptomycin or gentamicin owing to low uptake of these highly polar

molecules. High-level resistance results from the acquisition of aminoglycoside-modifying enzymes or, for streptomycin,

can result from ribosomal mutations that result in altered target binding. Resistance to the glycopeptide vancomycin

occurs through a well-characterized mechanism of reduced vancomycin-binding affinity, involving alterations in the

peptidoglycan synthesis pathway. Resistance ofEnterococcus spp.to the streptogramin quinupristindalfopristin (QD)

involves several pathways, including drug modification (by virginiamycin acetyltransferase (Vat)), drug inactivation

(through virginiamycin B lysase (Vgb)) and drug efflux (via the ATP-binding cassette protein macrolidestreptograminresistance protein (MsrC)). Resistance to the oxazolidinone linezolid is rare, but the most common pathway involves

mutation in the 23S ribosomal RNA ribosome-binding site. Resistance ofE. faecalis to the lipopetide daptomycin has

been shown to involve altered interactions with the cell membrane and requires the membrane protein LiaF and enzymes

involved in phospholipid metabolism, such as a member of the glycerophosphoryl diester phosphodiesterase family

(GdpD) and cardiolipin synthase (Cls).

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StreptograminsNaturally occurring antibiotics

that are produced by soil

bacteria and inhibit protein

synthesis. A mixture of the

streptogramins quinupristin

and dalfopristin is currently

available for clinical use and is

approved by the US Food and

Drug Administration for the

treatment of vancomycin-

resistant Enterococcus faecium

infections.

However, the occurrence of acquire high-level resist-ance to all available aminoglycosies eliminates thepotential for synergistic treatments. High-level resist-ance to streptomycin results from ribosomal mutations,which usually change the S12 ribosomal protein, or theacquisition of an aminoglycosie nucleotiyltransferase,ANT(3)-Ia117 or ANT(6)-Ia118. High-level resistance togentamicin is usually the consequence of a transposonthat encoes the bifunctional aminoglycosie-moifyingenzyme AAC(6)-IeAPH(2)-Ia, which confers resist-ance to all commercially available aminoglycosies exceptstreptomycin. Aminoglycosie-moifying enzymes cata-lyse the covalent moification of amino an hyroxylgroups within the aminoglycosie molecule, markelyecreasing the bining affinity between the antibiotican the bacterial ribosome119. E. faecium is intrinsicallyresistant to synergistic treatments using tobramycin owingto the ubiquitous AAC(6) (which confers resistance totobramycin)116, an a 16S ribosomal RNA methyltrans-ferase enzyme, EfmM, has also recently been linke tointrinsic aminoglycosie resistance in this species120.

Glycopeptide resistance. In the past, vancomycin was theusual alternative to ampicillin for infections cause byampicillin-resistant enterococci or in patients with severeallergy to -lactams. The emergence of high-level resist-ance to glycopepties in enterococci in 1986 (REFS 121,122)was surprising, as vancomycin ha been use for ecaeswithout the emergence of resistance. However, it is nowclear that vancomycin resistance is common in nature123.Inee, the members of several Gram-positive genera,such as Lactobacillus, Leuconostoc an Pediococcus124,are inherently resistant to glycopepties, as these organ-isms prouce peptioglycan precursors with ecreaseaffinity for vancomycin; a similar resistance is seen inEnterococcus gallinarum an Enterococcus casseliflavus.However, the actual origin of the genes responsible forhigh-level vancomycin resistance in enterococci has beenlinke to soil Paenibacillusspp.125.

The mechanism of vancomycin resistance has beenextensively reviewe previously126,127. Glycopeptie resist-ance involves two pathways: replacement of the terminald-Ala of peptioglycan precursors with d-lactate (d-Lac),which prouces high-level resistance, or with d-Ser, whichprouces low-level resistance; an prevention or estruc-tion of precursors that en in d-Alad-Ala by specificd,d-ipeptiases an carboxypeptiases. The d-Lacreplacement eliminates one of the five hyrogen bons

that woul be establishe between vancomycin an thed-Alad-Ala termini, resulting in an almost 1,000-folecrease in affinity127. Replacement of d-Ala by d-Seralso ecreases the bining affinity of glycopepties forpeptioglycan precursors, although to a lesser egree.Enterococcal strains with inuce or constitutivelyexpresse vancomycin resistance operons have reucefitness in vitro an ecrease issemination among micecompare with uninuce isogenic strains128.

Resistance to quinupristindalfopristin. Quinupristinalfopristin was the first rug approve by the US Fooan Drug Aministration (FDA) for the treatment

of VRE infections129. This antibiotic is a mix of twosemisynthetic streptogramins. Like the macrolies anlincosamies, quinupristinalfopristin inhibits proteinsynthesis by interacting with the 50S ribosomal subunitan prouces a synergistic effect by irect interactionof both compouns with a single nucleotie (A2062) atthe peptiyltransferase centre, leaing to a significantconformational change that results in bactericial activ-ity130. Most E. faecalis isolates are resistant to alfopristinowing to the activity of Lsa, a preicte ATP-biningprotein131. Several resistance mechanisms reuce theactivity of quinupristinalfopristin against E. faecium,incluing rug moification, inactivation an effluxthrough virginiamycin acetyltransferase (Vat)(virginia-mycin is another streptogramin mixture that is similarto quinupristinalfopristin), virginiamycin B lyase(Vgb)an the ATP-bining cassette protein macroliestreptogramin resistance protein (MsrC), respectively132,133.The erm genes, encoing enzymes that methylate 23SrRNA an cause resistance to macrolies, lincosamiesan quinupristin (the MLS

Bphenotype; quinupristin is a

streptogramin B antibiotic), result in ecrease bacteri-cial activity of quinupristinalfopristin in experimentalenocaritis129.

Linezolid resistance. Linezoli is the secon of the twocompouns that are approve by the FDA to treat VREan is use worlwie. This oxazoliinone is bacte-riostatic an inhibits protein synthesis by interferingwith the placement of the aminoacyl tRNA at the A siteof the bacterial ribosome134,135. Resistance to linezoliis still rare but has been ocumente in enterococcaloutbreaks an even sporaically in patients who havenever receive the antibiotic. The most commonmechanism of resistance foun in enterococci involvesG2576T mutations in genes encoing omain V of the23S rRNA (position 2576 refers to the nucleotie posi-tion originally assigne to rRNA genes in Escherichiacoli). This mutation interferes with the positioning ofcrucial nucleoties in the linezoli-bining site134. AsE. faecium an E. faecalis have six an four copies of therRNA genes, respectively, the levels of resistance corre-late with the number of mutate alleles135. Intracellularrecombination between gene copies also seems to havea role in the amplification of mutate rRNA genes, asbacteria that are eficient in recombinase A exhibita elay in such amplification136,137. Interestingly, thelinezoli resistance that is foun in some clinical iso-

lates of staphylococci, meiate by the acquire gene cfr(which encoes a methyltransferase that moifies posi-tion A2503 of the 23S rRNA), may have been transferrefrom an Enterococcus species138.

Daptomycin resistance. Daptomycin, a lipopeptie anti-biotic, has potent in vitro bactericial activity againstenterococci (incluing ampicillin- an vancomycin-resistant E. faecium) an is commonly use in theUnite States an other countries to treat VRE infec-tions (although it is not approve by the FDA for thisinication). Recently, whole-genome sequencing of aaptomycin-susceptible an a aptomycin-resistant

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E. faecalis clinical isolate recovere from a patient beforean after aptomycin therapy, respectively, showe thatsimultaneous mutations in two genes are sufficient tocause resistance139. One gene, mutation of which appearsto be essential for resistance to occur, encoes a mem-brane protein, LiaF, that is preicte to be a memberof a three-component regulatory system (LiaFSR)involve in the cell envelope stress-sensing response toantibiotics. The secon gene encoes a protein of theglycerophosphoryl iester phosphoiesterase family anprobably has a role in metabolism of membrane phos-pholipis139. Comparative analysis of the resistant ansusceptible isolates reveale that the resistant mutanthas alterations in the ultrastructure of the cell mem-brane an cell wall, an that aptomycin is less able toepolarize the cell membrane of the resistant isolate thanthat of the susceptible isolate. In vitro selection stuies ofthe sequence isolate E. faecalis str. V583 have suggestea role in aptomycin resistance for genes encoingcariolipin synthase, a phospholipi synthesis enzyme140.

ConclusionsThe enterococci are perhaps the most striking examplesof organisms that, historically, were regare as secon-rate pathogens (when compare with Staphylococcusaureus, group A streptococci an pneumococci, forexample) but which, over the past few ecaes, havebecome one of our most challenging nosocomial prob-lems. This change in status is in part ue to the increaseuse of antibiotics in hospitals worlwie: antibioticscan cause changes in the gut microbiota of patients,an these changes cause subsequent alterations in thelocal immune system. Enterococci take avantage ofthis phenomenon an conquer the prize niche of theGI tract, an this niche is the primary source of organ-isms that then cause enterococcal infections. Althoughhospital-associate clones ofE. faecium are intrinsicallyifferent in their core genome from strains in the com-munity-associate clae, the malleability of enterococcalgenomes has almost certainly contribute to the rise ofthese organisms as important nosocomial pathogens.Thus, the anticipate avances in enterococcal genom-ics shoul inclue the iscovery of important clues aboutthe evolution of enterococci an the elineation of the

important ifferences between community-associatean nosocomial enterococci, as well as ifferences in thenosocomial clae. In vivo screening for the activation ofspecific genes in ifferent moels of infection141 an forthe role of small RNAs142, non-coing RNA an anti-sense RNA143 is one of the approaches that may provieclues to unerstaning these ifferences.

Within the genus Enterococcus, E. faecium hasemerge as the most therapeutically challenging organ-ism. In this species, many of the ifferences betweenhospital- an community-preominant clones aroselong before the moern-ay hospital existe34. However,acquisition of antibiotic resistance genes, an probablyother genes, has escalate in recent years. At least inE. faecalis, this acquisition has occurre most signifi-cantly in lineages that lack the CRISPR efence systemagainst incoming foreign DNA, an these are the verylineages that contain the majority of hospital-associatestrains. The selective avantage conferre by antibioticresistance can be enormous, particularly in the hospitalsetting, where there is such wiesprea antibiotic use.

Because enterococci have shown the ability toevelop resistance to essentially every antibiotic useagainst them, novel approaches are neee. Futurestuies shoul further issect the mechanism by whichenterococci can colonize the GI tract an shoul thenfocus on ways to reuce or prevent this colonization.Strategies, incluing immunological approaches, toprevent infection in the face of colonization shoul beexplore. In aition, the eluciation of mechanismsof resistance to the newer anti-enterococcal antibioticssuch as aptomycin shoul yiel important insightsfor the evelopment of future therapeutic options.Aitional new strategies, perhaps phage-base ora combine therapeuticimmunological approach,shoul also be consiere.

For the foreseeable future, however, the enterococciwill remain an important nosocomial problem. Thus,continue stuy of enterococcal biology an geneticsis warrante an will unoubtely contribute to ourunerstaning of bacterial evolution, as well as yielinsights into the complex interactions between the hostan the resient bacterial flora that is a common sourceof nosocomial infections.

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AcknowledgementsThe authors have been supported by US National Institutesof Health grants R01 AI067861 and R37 AI47923 (B.E.M.),

and R00 AI72961 and R01 AI093749 (C.A.A.) from the USNational Institute of Allergy and Infectious Diseases (NIAID).The authors are grateful to J. Sillanpaa and S. Nallapareddyfor discussions and insights.

Competing interests statementThe authors declare no competing fi

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