Virulence and Antibiotic Resistance of Enterococci Isolated from Healthy Breastfed Infants

Abstract

Pathogenic ability has been extensively studied in clinical enterococci, but to a lesser extent in community-derived ones. Most studies to date in enterococci from healthy infants have been focused on Enterococcus faecalis, despite the growing concern about nosocomial infections caused by E. faecium. In this work, we studied the antibiotic resistance and virulence determinants of 26 E. faecalis and 15 E. faecium intestinal isolates from Spanish healthy breastfed infants. Overall, commensal enterococci studied contained antibiotic resistance and virulence genes, although their patterns were not according to those described for antibiotic-resistant hospital-associated enterococci. None of the isolates was resistant to vancomycin, although the majority showed resistance to some antibiotics. E. faecalis isolates harbored considerably more virulence determinants than E. faecium isolates, but some genes linked to colonization were abundant in both species. Hemolysin activity was not detected in any of the isolates; and the gelatinase gene, when present, was silent in E. faecium, whereas gelatinase activity occurred in half of the E. faecalis isolates studied. These results suggest an ambivalent role of some virulence determinants as elements of pathogenesis.

Introduction

Enterococci are commensal bacteria commonly found in human intestinal microbiota¹ and among the first colonizers of the intestines of a newborn.² Colostrum and breast milk are considered important sources of these bacteria.^3,4 Enterococcus faecalis and E. faecium are the most common species isolated from the human gut.⁵ In addition to their role in well-being and health as part of the commensal gut microbiota, enterococci have been safely used for decades in fermented foods and as probiotics for humans and farm animals.⁶ On the contrary, hospital environment has selected for traits that make colonization and transmission more likely, becoming the enterococci nosocomial opportunistic pathogens.⁷ However, the fraction of human-associated enterococci causing infection is infinitesimally small and in fact, native enterococcal factors related to virulence in commensal strains, are thought to play an adaptive role and contribute to bacterial survival and proliferation in their natural habitats.⁷ The excessive widespread use of antibiotics has increased the number of resistances in clinical and also in commensal bacteria.^8,9 A rapid increase in enterococci as agents of nosocomial infections has actually been observed,¹⁰ most of them ascribed to E. faecalis. However, E. faecium is an emergent and challenging nosocomial problem.^6,10 This shift in species is of paramount clinical importance, as E. faecium is by far the more difficult of the two species to treat.⁶ Pathogenic E. faecium seems to be associated to a genetically related epidemic genogroup of vancomycin-resistant organisms particularly widespread in hospitals.^7,11,12

The emergence of resistance to various antimicrobial agents, especially vancomycin, has become a major clinical and epidemiological threat especially in neonates. The risk of death from vancomycin-sensitive enterococci is 45% and increases to 75% for those infected with vancomycin-resistant enterococci (VRE) strains.¹³ Moreover, the variable traits that have converged in hospital-adapted multidrug-resistant enterococci include a number of genetic virulence factors related with cytolysin (cylM, cylB, and cylA), gelatinase (gelE), hyaluronidase (hyl), aggregation substance (agg₂), and cell wall adhesins (esp), the significance of which has been concisely reviewed by others.^6,7 Acquired elements are not the only factors behind the recent success of hospital-associated E. faecium, and divergence between pathogenic and commensal clades took place many years ago.¹² Virulent hospital enterococci seem to be less well adapted for life outside their environment,¹⁴ and these virulent clades are replaced with time after patients leave the hospital.¹⁵

In this work, we studied the antibiotic resistance profiles and the presence of virulence determinants in E. faecalis and E. faecium isolated from healthy breastfed infants. The objective was to obtain further data about the distribution of potential pathogenic traits in community-derived intestinal isolates, seeing the double role of both species as commensal and potential pathogenic bacteria.

Materials and Methods

Isolation and identification of gut enterococci

Fecal samples were collected from 21 Spanish breastfed infants younger than 6 months. The samples were kept at 4°C and processed within the 12 hrs of collection. All volunteers fulfilled the following criteria: (i) healthy infants and women who had not been prescribed antibiotics for at least 3 months before the study; (ii) normal pregnancy; and (iii) absence of infant and/or maternal perinatal problems. Explicit informed oral consent was obtained from the parents of the infants who provided the samples used in this study.

Samples were homogenized and dilutions were plated on De Man, Rogosa and Sharpe (MRS) agar (Biolife, Milano, Italy) and incubated at 37°C for 24–48 hr. Three to five colonies per sample were streaked on Kenner-Fecal agar (Biolife) for selection and the isolates subjected to Gram staining, catalase test, and cell morphology examination.

All the isolates were stored at −80°C in MRS broth containing 10% (v/v) glycerol (Panreac, Barcelona, Spain) and propagated in MRS broth twice before being used in experiments.

Potential enterococci were identified by using 16S rRNA gene sequencing¹⁶ and the sequences were compared with type strains included in Ribosome Database Project (http://rdp.cme.msu.edu). Moreover, primers proposed by Layton et al.⁵ were used to differentiate between species of Enterococcus (Table 1).

Table 1.

Primers Used in This Study

Gene	Primers	Sequence (5′ to 3′)	Product size (bp)	References
16S rRNA genes
	63F	CAGGCCTAACACATGCAA GTC	1,300	¹⁶
	1387R	GGG CGG WGT GTA CAA GGC
Enterococcus faecium/durans/hirae
	DU1B	ACTGATATTAAGACAGCGGTACAA	286	⁵
	DU2B	AGATAGGTGTTTGGCCTGTCAT
	FM1B	ACAATAGAAGAATTATTATCTG	214	⁵
	FM2B	CGGCTGCTTTTTTGAATTCTTCT
	HI1	CTTTCTGATATGGATGCTGTC	186	⁵
	HI2	AAATTCTTCCTTAAATGTTGC
Vancomycin resistance genes
vanA	VAN-AF	TCTGCAATAGAGATAGCCGC	377	¹⁸
	VAN-AR	GGAGTAGCTATCCCAGCATT
vanB	VAN-BF	GCTCCGCAGCCTGCATGGACA	529	¹⁸
	VAN-BR	ACGATGCCGCCATCCTCCTGC
Virulence factors
agg2	TE32	GTTGTTTTAGCAATGGGGTAT	1,210	²⁰
	TE33	CACTACTTGTAAATTCATAGA
cpd	TE51	TGGTGGGTTATTTTTCAATTC	782	²¹
	TE52	TACGGCTCTGGCTTACTA
cob	TE49	AACATTCAGCAAACAAAGC	1,405	²¹
	TE50	TTGTCATAAAGAGTGGTCAT
ccf	TE53	GGGAATTGAGTAGTGAAGAAG	543	²¹
	TE54	AGCCGCTAAAATCGGTAAAAT
cad	TE42a	TGCTTTGTCATTGACAATCCG	1,299	²⁰
	TE43a	ACTTTTTCCCAACCCCTCAA
cylM	TE13	CTGATGGAAAGAAGATAGTAT	742	²¹
	TE14	TGAGTTGGTCTGATTACATTT
cylB	TE15	ATTCCTACCTATGTTCTGTTA	8,430	²¹
	TE16	AATAAACTCTTCTTTTCCAAC
cylA	TE17	TGGATGATAGTGATAGGAAGT	517	²¹
	TE18	TCTACAGTAAATCTTTCGTCA
espfm	TE104	TTGCTAATGCAAGTCACGTCC	955	²⁰
	TE105	GCATCAACACTTGCATTACCGAA
espfs	TE34	TTGCTAATGCTAGTCCACGACC	933	²¹
	TE36	GCGTCAACACTTGCATTGCCGAA
efaAfm	TE37	AACAGATCCGCATGAATA	735	²¹
	TE38	CATTTCATCATCTGATAGTA
efaAfs	TE5	GACAGACCCTCACGAATA	705	²¹
	TE6	AGTTCATCATGCTGTAGTA
gelE	TE9	ACCCCGTATCATTGGTTT	419	²¹
	TE10	ACGCATTGCTTTTCCATC
hyl	HYL n1	ACAGAAGAGCTGCAGGAAATG	276	²²
	HYL n2	GACTGACGTCCAAGTTTCCAA

Virulence determinants: agg₂, aggregation substance (AS); cad, ccf, cob, and cpd, sex pheromones; cylM, cylB, and cylA, cytolysin biosynthesis; efaA_fs and efaA_fm, cell wall adhesins; esp_fs and esp_fm, enterococcal surface proteins; gelE, gelatinase; hyl, hyaluronidase.

Screening for antibiotic resistance and van genes

Antibiogram of enterococcus isolates was determined by the disc diffusion method according to Nueno-Palop and Narbad.¹⁷ The antibiotics tested were chloramphenicol (10 μg), erythromycin (15 μg), rifampicin (30 μg), tetracycline (10 μg), and vancomycin (30 μg) (Oxoid, Unipath Ltd., Basingstoke, United Kingdom). The presence of the vancomycin resistance genes vanA and vanB was determined by PCR with specific primers¹⁸ (Table 1).

Screening for gelatinase and hemolysin activities

Production of gelatinase was determined on Todd-Hewitt agar supplemented with 3% gelatin (Difco, Detroit, MI). Isolates were streaked onto plates and incubated at 37°C under anaerobic conditions for 24 hrs. Then plates were kept at 4°C for 5 hrs. Zones of turbidity around the colonies indicated hydrolysis of gelatin. Hemolysin activity was determined on blood agar base supplemented with 7% horse blood (Oxoid) after 48 hrs of incubation at 37°C. Zones of clearing around colonies indicated hemolysin production.

Screening for enterococcal virulence factors

Genomic DNA was isolated from overnight enterococcal cultures according to the method of Ruiz-Barba et al.¹⁹ Known virulence gene was detected by means of PCR amplifications with the specific primers listed in Table 1 and conditions described in their respective references.

Results

Isolation and species identification of breastfed infant gut Enterococcus

Samples from the 21 breastfed infants studied resulted in 41 Gram-positive and catalase-negative isolates that showed coccoid morphology in contrast-phase microscopy. Of the 41 isolates, 26 (from 15 infants) were identified as E. faecalis and 15 (from seven infants) as E. faecium by means of 16S rRNA gene sequencing and PCR with species-specific primers.

Antibiotic resistance and van genes

Determination of the antibiotic resistances on the strains tested yielded five different resistance profiles within each Enterococcus species (Table 2). The antibiotic profiles most commonly encountered, summed one or more resistance; profiles resistant to more than one antibiotic were less frequent.

Table 2.

Profiles of the Antibiotic Resistance of Enterococcus faecalis and Enterococcus faecium Strains Isolated from Breastfed Infant Feces

Profile	Isolates ^a	Infants ^b	VAN	RIF	TET	CHL	ERY
E. faecalis
Rfs1	1	1	S	S	R	R	R
Rfs2	2	1	S	S	R	R	S
Rfs3	1	1	S	S	R	S	R
Rfs4	16	9	S	S	R	S	S
Rfs5	6	4	S	S	S	S	S
Resistant isolates (%)			0.0	0.0	76.9	11.5	7.7
E. faecium
Rfm1	1	1	S	R	S	S	R
Rfm2	1	1	S	R	S	S	S
Rfm3	1	1	S	S	R	S	R
Rfm4	8	4	S	S	S	S	R
Rfm5	4	4	S	S	S	S	S
Resistant isolates (%)			0.0	13.3	6.7	0.0	66.7

Number of isolates with a given profile.

Number of infants in which isolates with a given profile were found.

S, sensitive; R, resistant; VAN, vancomycin; RIF, rifampicin, TET, tetracycline; CHL, chloramphenicol; ERY, erythromycin.

None of the isolates showed resistance to vancomycin and all of them were negative for van genes. Chloramphenicol and rifampicin resistance were scarce among enterococci, appearing only in two E. faecalis and two E. faecium isolates, respectively.

Tetracycline resistance was the most prevalent resistance trait among E. faecalis and rare in E. faecium, whereas erythromycin resistance was the most prevalent resistance trait among E. faecium isolates and unusual in E. faecalis.

Virulence determinants and hemolysin and gelatinase activities

Enterococcal isolates were examined for the presence of 16 genetic virulence determinants by means of PCR. The 26 E. faecalis isolates and the 15 E. faecium isolates resulted in 17 and 12 virulence profiles, respectively (Vfs and Vfm; Table 3). The E. faecalis profiles harbored much more virulence determinants than those of E. faecium mainly due to a higher detection of cylA, cylB, and cylM (cytolysin biosynthesis); agg₂ (AS); gelE (gelatinase); and cpd, cob, and cad (sex pheromone determinants) in E. faecalis. Cytolysin determinants were found in seven of the E. faecalis profiles, although none of enterococci showed a hemolysin activity under the conditions tested. In addition, E. faecalis profiles with cytolysin determinants exhibited AS and sex pheromone determinants.

Table 3.

Virulence-Determinant Profiles and Hydrolytic Activities Found in E. faecalis and E. faecium Strains Isolated from Breastfed Infant Feces

Profile	Isolates ^a	Infants ^b	agg₂	cad	ccf	cob	cpd	cylM	cylB	cylA	efaA_fm	efaA_fs	esp_fs	esp_fm	gelE	hyl	CYL	GEL	^c
E. faecalis
Vfs1	1	1	−	−	+	−	−	−	−	−	−	+	−	−	−	−	−	−
Vfs2	1	1	−	−	−	−	+	−	−	−	−	+	−	−	−	−	−	−
Vfs3	1	1	−	+	−	−	−	−	−	−	−	+	+	−	+	−	−	−
Vfs4	1	1	−	−	+	−	−	−	−	−	−	+	−	−	+	−	−	+	(1/1)
Vfs5	1	1	−	+	+	+	+	−	−	−	−	+	−	−	+	−	−	+	(1/1)
Vfs6	3	3	−	+	+	+	+	−	−	−	−	+	+	−	+	−	−	+	(2/3)
Vfs7	2	2	+	+	+	+	−	−	−	−	−	−	+	+	+	−	−	+	(1/2)
Vfs8	1	1	+	−	+	+	+	−	−	−	−	+	+	−	+	−	−	+	(1/1)
Vfs9	3	1	+	+	+	+	+	−	−	−	−	+	+	−	+	−	−	+	(3/3)
Vfs10	4	3	+	+	+	+	+	−	−	−	−	+	+	+	+	−	−	+	(1/4)
Vfs11	1	1	+	+	+	+	+	−	−	+	−	+	+	−	+	−	−	−
Vfs12	1	1	+	+	+	+	+	+	+	+	−	−	+	+	−	−	−	−
Vfs13	1	1	+	+	+	+	+	+	+	+	−	+	+	−	−	−	−	−
Vfs14	1	1	+	+	+	+	+	+	+	+	−	+	+	+	−	−	−	−
Vfs15	1	1	+	+	+	+	−	+	+	+	−	+	+	−	+	−	−	−
Vfs16	1	1	+	+	+	+	+	+	+	+	−	−	+	−	+	−	−	+	(1/1)
Vfs17	2	2	+	+	+	+	+	+	+	+	−	+	+	+	+	−	−	+	(1/2)
Positive isolates (%)			69.2	84.6	92.3	84.6	76.9	26.9	26.9	30.8	0.0	84.6	84.6	38.5	80.8	0.0	0.0	46.2
E. faecium
Vfm1	1	1	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−
Vfm2	1	1	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−
Vfm3	1	1	−	−	−	−	−	−	−	−	+	−	−	+	−	−	−	−
Vfm4	1	1	−	−	+	−	−	−	−	−	+	−	−	−	−	−	−	−
Vfm5	2	1	−	−	+	−	−	−	−	−	+	−	−	+	−	−	−	−
Vfm6	2	1	−	−	+	−	−	−	−	−	+	−	−	+	+	−	−	−
Vfm7	1	1	−	+	+	−	−	−	−	−	+	−	−	+	+	−	−	−
Vfm8	1	1	−	−	+	−	+	−	−	−	−	−	−	−	+	−	−	−
Vfm9	1	1	−	+	+	−	+	−	−	−	+	−	−	−	+	−	−	−
Vfm10	1	1	−	+	+	−	+	−	−	−	+	−	−	+	+	−	−	−
Vfm11	2	2	+	−	+	−	−	−	−	−	+	−	−	+	+	−	−	−
Vfm12	1	1	+	−	+	−	+	−	−	−	+	+	−	+	+	−	−	−
Positive isolates (%)			20.0	20.0	80.0	0.0	15.4	0.0	0.0	0.0	86.7	6.7	0.0	66.7	60.0	6.7	0.0	0.0

Number of isolates with a given profile.

Number of infants in which isolates with a given profile were found.

(Number of gelatinase-positive isolates/total of isolates within a given virulence profile).

Virulence determinants: agg₂, aggregation substance (AS); cad, ccf, cob, and cpd, sex pheromones; cylM, cylB, and cylA, cytolysin biosynthesis; efaA_fs and efaA_fm, cell wall adhesins; esp_fs and esp_fm, enterococcal surface proteins; gelE, gelatinase; hyl, hyaluronidase. Hydrolytic activities: CYL, hemolysin activity; GEL, gelatinase activity.

The gelE gene was found in both species, although the gelatinase activity was only detected in 12 out of 21 gelE⁺ E. faecalis isolates, and was not detected in any of the 9 gelE⁺ E. faecium isolates. The adhesion gene esp was detected in high proportion in the enterococci studied. However, in E. faecium, only esp_fm was detected, whereas simultaneous esp_fs and esp_fm amplification was evidenced in several E. faecalis profiles. In addition, most enterococci were positive to their species-specific efaA genes.

Discussion

E. faecalis and E. faecium were the enterococcal species most frequently detected in infant and neonatal fecal samples.^20,21 In accordance with this, those were the two species identified in this study among the enterococci isolated from healthy breastfed infants.

This study provides a snapshot view of the antibiotic resistance and virulence determinants present in community enterococci isolated from healthy Spanish breastfed infants. Pathogenic potential has also been studied in commensal E. faecium, which is often excluded. It is of note that the studied strains were susceptible to many of the antibiotics tested, and few of them showed more than one antibiotic resistance. Furthermore, they did not show vancomycin resistance and none of them harbored the vanA or vanB genetic determinants. Although VRE are a major threat in hospital environments, isolation from healthy individuals rarely occurs.^22,23

Chloramphenicol resistance was scarce among our isolates, although it was detected in two E. faecalis isolates (Table 2). This resistance is considered transmissible and increasing among VRE, compromising the therapeutic options for bloodstream infections.²⁴ Differences in resistance to erythromycin and tetracycline between the two enterococcal species were evidenced, since high incidence of tetracycline resistance, but low incidence of erythromycin in E. faecalis, and the opposite in E. faecium were observed. Similar results have been previously reported in E. faecalis isolated from healthy Norwegian infants²² and in E. faecium and/or E. faecalis isolates from clinical²⁵ or food samples.²⁶ However, both resistances occurred simultaneously in two E. faecalis and one E. faecium of our isolates. Jiménez et al.²³ also observed both tetracycline and chloramphenicol resistance in 15 E. faecalis and 9 E. faecium isolated from animal and human milk, highlighting its importance as possible source of resistant enterococci.

In addition, enterococcal isolates were examined in this work for the presence of 16 putative virulence genes and cytolysin and gelatinase activities. Globally, E. faecalis isolates harbored significantly more virulence determinants than E. faecium isolates, in concordance with previous data.^25–27 Most of the E. faecalis virulence profiles (Table 3) carried between 6 and 12 virulence determinants, which is according with the usual genotype found in this species.¹⁷ In contrast, E. faecium profiles showed fewer than seven virulence determinants.

The main virulence trait seems to be cytolysin that lyses eukaryotic cells and enhances the virulence of pathogenic strains in animal models.²⁸ E. faecium isolates assayed did not show cylM, B, and A according to others.^23,29 These determinants were only encountered in 7 out of 26 E. faecalis isolates, although none of them showed a hemolysin activity under the conditions tested. This result contrasts with the 9 out of 31 hemolytic E. faecalis isolates found in healthy Norwegian infants.²² The lack of hemolytic activity in enterococci harboring cylM, B, and A genes has been attributed to the lack of structural genes (cylL _l and cylL _s ) or intrinsic or environmental factors affecting their expression.^30,31 Enterococcus isolates harboring cyl determinants, but not expressing the hemolytic phenotype, have been also observed.^27,32

In the same way, gelatinase determinant (gelE) was extensively present in both E. faecium and E. faecalis isolates (60% and 81%, respectively) in agreement with results of Lopes et al.³³ and Solheim et al.²² with E. faecalis from healthy infants. Gelatinase activity was found in half of the gelE⁺ E. faecalis. The negative gelatinase phenotype could be explained by the absence of the regulator,³⁴ silent gelE genes activated by certain exogenous factors,²⁷ cell density-dependent regulation,³⁵ or even a loss of activity during freezing conservation in the laboratory.³³ Gelatinase production is not exclusive of the species associated with infections, but rather is also an enzyme able to degrade casein and therefore is common in dairy enterococci isolated from milk,³³ and might be adaptive and linked to breastfeeding in neonates.

Another major virulence trait is AS, a component of the pheromone-responsive plasmid exchange system involved in several stages of the enteroccocal infection.³⁶ Agg₂ was found in 69% of E. faecalis tested, in agreement with results of Solheim et al.²² and in three E. faecium isolates. Although agg₂ has been reported in E. faecium strains of food and clinical origin,³⁷ this virulence factor has been described mostly in E. faecalis.²⁷

Some enterococci have highly effective gene transfer mechanisms. Virulence genes encoding cytolysin, gelatinase, and AS are known to be associated with certain pheromone-responsive plasmids.³⁸ E. faecalis isolates in this work showed a higher incidence of sex pheromone determinants (cpd, ccf, cad, and cob) than E. faecium ones. Cytolysin and AS determinants could be associated with the presence of sex pheromone determinants in our isolates. A lower prevalence of cytolysin compared to AS in E. faecalis was observed in agreement with Coque et al.³⁹ We found ccf to be the only sex pheromone determinant present in E. faecium at levels comparable with E. faecalis, in agreement with findings from neonates.²¹ In contrast, it was not detected in E. faecium isolates from breast milk,²⁹ nor from food or human clinical infections.²⁷

Genes for enterococcal surface protein and cell wall adhesins (esp_fm, esp_fs, efaA_fm, and efaA_fs) were frequent in their correspondent species in accordance with Togay et al.³⁷ Surface protein (esp) has been linked to a putative enterococcal pathogenicity island, which is different in E. faecalis and E. faecium.⁴⁰ In the case of E. faecalis, an 85% of positive scores were observed in accordance with the esp frequencies reported in isolates from healthy infants.²² The presence of esp was quite common among our E. faecium isolates (67%) in contrast with the absence of this gene previously described in breast milk isolates.²⁹ The adhesion genes efaA_fm/efaA_fs were present in about 85% of our isolates in concordance with previous studies on isolates from colostrum and breast milk.^4,29 Furthermore, efaA_fm has been found in 100% of starter E. faecium strains with a long record of safe use in food.²⁷ In contrast to efaA_fs of E. faecalis, the role of adhesin efaA_fm in virulence has not yet been demonstrated.⁴¹

As for the production of gelatinase, the capacity to degrade hyaluronic acid may benefit the colonization process. Hyaluronidase gene has been described mainly in clinical isolates and linked to esp.⁴² In accordance, only one E. faecium isolate of the commensal enterococci studied was positive for this trait.

The findings of our study show that antibiotic resistance and virulence determinants were present to a certain extent in enterococci isolated from healthy breastfed infants, particularly in E. faecalis, but also in E. faecium, for which very limited studies had been previously carried on in community-based isolates. Both species analyzed were classified according to their different antibiotic resistance profiles and virulence-determinant profiles obtained. Most common antibiotic profiles (Rfs 4, Rfs 5, Rfm 4, and Rfm 5) grouped the majority of isolates, and hence, the infants analyzed. Those profiles showed that commensal enterococci were susceptible to majority of the antibiotics tested and all the isolates analyzed were clear of vancomycin resistance typical of clinical isolates.

Profiles corresponding to virulence genes showed high variability for both species, but more virulence determinants were present in E. faecalis than in E. faecium. However, all the isolates were clear of vancomycin resistance and hemolysin activity typical of many clinical isolates. The presence of antibiotic resistance and virulence determinants in enterococci isolated from healthy infants points out the multifactorial and complex nature of enterococcal pathogenesis, in which the host immunological status may also play a key role. The abundance of determinants linked to colonization among our E. faecium isolates might contribute to explain the ability described in clade B strains to proliferate and replace antibiotic- resistant hospital-associated clade A E. faecium once patients leave the hospital.^14,15 Further investigations, including more strains representing infant diversity from different countries and personal conditions, are needed to understand the role of enterococci as opportunistic pathogens, along with their beneficial one in the colonization of infant gut.

Footnotes

Acknowledgment

This work was supported by project RTA2013-00029-00-00 from the Spanish Ministry of Economy and Competitiveness (MINECO).

Disclosure Statement

No competing financial interests exist.

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