Introduction

Enterococci are part of the normal human microbial flora. Historically, the majority of invasive enterococcal infections were caused by Enterococcus faecalis, followed by Enterococcus faecium [1]. In recent decades, however, the epidemiology of invasive enterococcal infections appears to be changing worldwide, and a number of trends have been recognized, notably, the global emergence of enterococci as important nosocomial pathogens and the emergence of resistance to commonly used antimicrobial agents, including penicillins, aminoglycosides and glycopeptides [1].

An increase in the number of E. faecium strains in hospitals in different countries has been documented during the last decade [2-4]. These isolates had in common not only the antibiotic resistance traits (to ampicillin, quinolones and to glycopeptides in some cases) but also several virulence factors that might have contributed to the success of E. faecium as a leading nosocomial pathogen [4,5]. Although these strains were initially classified within a single clonal complex 17, it appears that the genetic diversity of this CC allows the classification of all isolates in three main lineages (17, 18, and 78), which is a more accurate representation of the recent evolution of these isolates[6].

Management of severe infections due to resistant enterococcal strains, especially E. faecium, has therefore become a therapeutical challenge. However, most of the reported experiences regarding enterococcal infections concern the general, non-immunocompromised population, and they mainly involve vancomycin-resistant strains [7-12]. Additionally, the majority of the studies published to date have been carried out in the United States, where the epidemiological situation is very different from that occurring in Europe [1,13]. Furthermore, information regarding bloodstream infection (BSI) due to E. faecium in immunosuppressed patients with cancer is particularly scarce [10,11,14,15]. Given the above, the aim of the present study was to describe the incidence and risk factors for vancomycin-susceptible E. faecium BSI in a large prospective cohort of cancer patients. We also aimed to ascertain the clinical features, antimicrobial susceptibility, genotypes and outcome of BSI due to E. faecium in this population.

Materials and Methods

Results

During the study period 1287 consecutive episodes of BSI were recorded. Of the 550 (42.5%) episodes caused by Gram-positive bacteria, 105 were due to enterococci (19%). Thirteen episodes of enterococcal BSI were not included in the study because they were caused by species other than E. faecium or E. faecalis (E. gallinarum 6, E. casseliflavus 2, E. avium 2, E. hirae 1, E. durans and E. raffinosus 1). Thus, 54 episodes of BSI caused by E. faecium and 38 by E. faecalis were finally included in the study. Four patients with two episodes of enterococcal BSI were included since they were considered to present different episodes, separated by at least four weeks.

The incidence of E. faecium BSI increased significantly over time (22 episodes/126610 admissions from 2006 to 2009 vs 32 episodes/80586 admissions from 2010 to September 2012; p=0.002). By contrast, the incidence of E. faecalis BSI remained stable over time (p=0.215).

Table 1 shows the baseline and clinical characteristics of patients with enterococcal BSI compared by groups. Patients with BSI due to E. faecium were more likely to have received previous antibiotics (mainly carbapenems), previous antifungal prophylaxis and previous blood transfusion. Likewise, they had more prolonged neutropenia than did patients with E. faecalis BSI, and were more likely to have a concomitant infection. In addition, there was a trend in the former group of patients towards haematological malignancy as the most frequent underlying disease, and for there to be a venous catheter in place. The most frequent origin of BSI was an endogenous source in 38% of cases (41% in the E. faecium group vs 34% in the E. faecalis group), followed by catheter infection in 15% of cases (15% vs 16%, respectively) and cholangitis in 13% (15% vs 10.5%, respectively). BSI originating in the urinary tract tended to be more frequent in the group of E. faecalis, whereas neutropenic enterocolitis tended to be more frequent in the E. faecium group. After applying a logistic regression model the only variable found to be an independent risk factors for E. faecium acquisition was previous use of carbapenems (OR 10.24; 95% CI, 1.35-77.66).

Microbiological studies

All E. faecium isolates were vancomycin and teicoplanin susceptible. Only two strains (2.9%) of the 54 isolates were ampicillin susceptible with MICs ≤1 µg/mL. During the study period 97.1% of strains showed high-level resistance to ampicillin, and the rates of high-level resistance to streptomycin and gentamycin were 74.6% and 32.8%, respectively. Quinolone resistance accounted for 88.8% of the isolates, with MICs ≥4 µg/mL.

Among 30 ampicillin-resistant E. faecium strains available for genotyping, 13 PFGE patterns were obtained corresponding to 7 STs (Figure 1). ST117 was dominant, accounting for 16 isolates (53.3%) belonging to PFGE type D (n=10; 62.5%) and PFGE type A (n=6; 37.5%). Five isolates (16.7%) belonged to ST17 of PFGE type B (n=4) and H (n=1), three isolates (10%) belonged to ST 78, two (6.7%), to ST18, two (6.7%) to ST203 and one to ST192. A single isolate belonged to a new ST844 (Table S1). Considering the new classification recently proposed by Willems and co-workers, 5 isolates belonged to lineage 17 (ST17), two isolates belonged to lineage 18 (ST18) and 23 isolates belonged to lineage 78 (ST78, ST117, ST192, ST203 and ST844) [6]. From 2006 to 2009, one isolate out of eight available for genotyping belonged to ST117 (year 2009). In contrast, from 2010 to 2012, 15 out of 22 isolates belonged to ST117, with PFGE patterns D (n=10) and A (n=5). The allelic profiles for all different STs are detailed in Table S1.

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Figure 1. Cluster analysis of Pulsed-field Gel Electrophoresis (PFGE) SmaI macrorestriction fragments of the 30 Enterococcus faecium strains followed by multilocus sequence typing (MLST) data.

MLST was inferred in those strains that were marked with an asterisk. For dendrogram construction, optimization and band position tolerance were set at 0.5% and 1.0% respectively. The cut-off value to define the PFGE patterns was set at 80% similarity.

https://doi.org/10.1371/journal.pone.0074734.g001

Antibiotic treatment and patient outcomes are detailed in Table 2. The large majority of patients received empirical antibiotic therapy (91%). The most frequently antibiotic used was a glycopeptide (42% of cases), followed by third- and fourth-generation cephalosporins (29%), β-lactam plus β-lactam inhibitors (26%) and carbapenems (25%). More patients with E. faecalis BSI received a combination of a β-lactam plus β-lactam inhibitor empirically, whereas more patients in the E. faecium group were given a glycopeptide. Patients with E. faecium BSI were more likely to have received inadequate initial empirical antibiotic therapy than were patients with E. faecalis BSI, and time to adequate empirical antibiotic therapy was also longer in the former group. No significant differences were found between the two groups regarding other outcomes such as early and overall mortality rates.

	E. faecalis	E. faecium	p
	n=38 (%)	n=54 (%)
Empirical antibiotic therapy	35 (92)	49 (91)	1.00
Inadequate empirical antibiotic therapy	9 (24)	24 (44)	0.041
β-lactam + β-lactam inhibitor	13 (37)	9 (18)	0.054
Cephalosporin	11 (31)	13 (26.5)	0.63
Carbapenem	8 (23)	13 (26.5)	0.80
Glycopeptide	10 (29)	25 (51)	0.046
Quinolone	1 (3)	2 (4)	1.00
Aminoglycoside	5 (14)	5 (10)	0.73
Combination therapy	8 (23)	6 (12)	0.24
Days to adequate empirical antibiotic therapy (median, range)	0 (0-6)	1 (0-4)	0.036
Intensive care unit admission	5 (13)	7 (13)	1.00
Mechanical ventilation	2 (6)	4 (8)	1.00
Overall case-fatality rate (30 days)	10 (26)	16 (30)	0.72
Early case-fatality rate (48 hours)	1 (3)	3 (6)	0.64

Table 2. Antibiotic therapy and outcome of patients with enterococal bacteraemia compared by groups.

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Risk factors associated with overall case-fatality in the cohort of patients with enterococcal BSI are shown in Table 3. The use of current corticosteroids, shock at presentation, intensive care unit (ICU) admission, mechanical ventilation and unknown source of BSI were the variables most frequently found in the group of patients who died. However, after applying a logistic regression model the only variables found to be independent risk factors for overall case-fatality were the current use of corticosteroids (OR 4.18; 95% CI, 1.34-13.01) and ICU admission (OR 9.97; 95% CI, 1.96-50.63). BSI due to E. faecium was not identified as a risk factor for overall mortality.

	E. faecalis	E. faecium	p	Adjusted OR	p
Characteristic	n=38 (%)	n=54 (%)		(95%CI)
Male sex	43 (65)	16 (61.5)	0.81	1.23 (0.38-3.89)	0.72
Age (yrs, median, range)	61 (26-78)	59 (21-83)	0.15	0.99 (0.95-1.03	0.65
Underlying haematological disease	49 (74)	16 (61.5)	0.30
Neutropenia (<500)	40 (61)	14 (54)	0.64
Corticosteroids	20 (30)	17 (65)	0.004	4.18 (1.34-13.01)	0.013
Shock at presentation	1 (1.5)	5 (19)	0.006	3.43 (0.27-43.70)	0.34
Inadequate empirical antibiotic therapy	26 (39)	7 (27)	0.33
Intensive care unit admission	3 (4.5)	9 (35)	<.001	9.97 (1.96-50.63)	0.006
Mechanical ventilation	1 (2)	5 (22)	0.005
Unknown source of bacteraemia	3 (4.5)	5 (19)	0.038	3.99 (0.68-23.40)	0.12
E. faecium bacteraemia	38 (58)	16 (61.5)	0.81

Table 3. Risk factors for overall mortality (30 days) in the cohort of patients with enterococcal bacteraemia.

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Discussion

We observed a dramatic increase in the incidence of ampicillin-resistant, vancomycin-susceptible E. faecium BSI in patients with cancer over the study period. Historically, E. faecalis was the responsible for the large majority of all clinical enterococcal infections, with E. faecium being less frequently isolated. However, in the late 1990s the ratio of E. faecalis to E. faecium infections in the United States shifted in favour of E. faecium, while in Europe the first reports of increased numbers of infections due to E. faecium were published in the mid-1990s [1,9,13]. Despite this, information regarding BSI caused by vancomycin-susceptible E. faecium in immunosuppressed cancer patients is scarce in the literature, and all the articles published to date are retrospective studies [11,14,15,22]. Thus, our study is the first prospective analysis of a cohort of cancer patients with vancomycin-susceptible E. faecium BSI to be conducted at a time when enterococcal infections are gaining importance worldwide, both in terms of dissemination and antimicrobial resistance. It is also the first study to describe how vancomycin-susceptible E. faecium outnumbers E. faecalis in this high-risk population of patients with BSI in our geographical area.

Although several studies have focused on risk factors for vancomycin-resistant enterococcal infection or colonization [23,24], little is known about risk factors for BSI due to vancomycin-susceptible E. faecium. In our study we identified previous use of carbapenems as the only independent risk factor associated with E. faecium BSI. Since the early research of Boyce et al., which related ampicillin-resistant enterococci to the use of imipenem, several authors have demonstrated the association of ampicillin-resistant enterococci with β-lactams [9-11,16,25]. Antibiotics may facilitate colonization and infection by depleting the gastrointestinal tract of its normal anaerobic flora and by selecting enterococci due to limited bactericidal activity against these organisms. The use of broad spectrum β-lactams (including carbapenems) in patients with cancer and frequently-associated febrile neutropenia is very common in clinical practice. Furthermore, the emergence of multidrug-resistant organisms (especially extended-spectrum β-lactamase-producing Enterobacteriaceae) among cancer patients in our centre often forces us to use carbapenems as the treatment of choice [26]. A judicious use of antibiotics is therefore needed in order to avoid the development and dissemination of bacterial resistance.

Our study shows that all the enterococci isolates remained susceptible to glycopeptides. Although vancomycin resistance has become an emerging health problem worldwide, it is less important in Europe than in the United States, and as yet it does not seem to be a problem in our geographical area [1,13]. However, the emergence of E. faecium is of potential concern as it is more commonly associated with vancomycin resistance than are the other enterococci [27]. The large majority of E. faecium isolates showed a high level of resistance to ampicillin, and only two strains were ampicillin susceptible. Notably, the two patients carrying the two susceptible strains had not received carbapenems previously.

PFGE and MLST analysis of 30 available E. faecium isolates showed that all isolates belonged to the three major recently described hospital-associated E. faecium lineages (17, 18 and 78) [6]. Of note, the ST117 (lineage 78) was especially frequent in the last three years, which may explain, at least partially, the emergence of E. faecium in our centre. This finding is in line with recent molecular epidemiological studies that have identified these three lineages (formerly CC17) as being responsible for the worldwide emergence of ampicillin-resistant E. faecium. These three lineages have adapted extremely well to the hospital environment, including the acquisition of ampicillin resistance and the esp virulence gene, which is associated with biofilm formation. Therefore, these lineages have become the leading cause of hospital-acquired E. faecium infections and outbreaks [2-4,28]. The partial replacement of ampicillin-susceptible E. faecalis by hospital-associated lineages of ampicillin-resistant E. faecium is worrying, since it may set the stage for the emergence of vancomycin-resistant E. faecium.

There is controversy in the literature regarding the association between E. faecium infection and mortality. Some authors have reported increased mortality in those patients with BSI due to ampicillin-resistant E. faecium [7,10,16]. However, it is still unclear if the increase in mortality actually depends on infection or, rather, whether infection behaves as a marker of the severity of underlying diseases [29]. In our study, and in line with some previous reports, we found no association between E. faecium BSI and increased mortality [8,9,14,28]. Interestingly, some in vitro studies have suggested that enterococcal virulence determinants are more frequently found in E. faecalis isolates than in E. faecium isolates [30]. On the other hand, some studies have reported that E. faecium is more often resistant to phagocytosis than is E. faecalis [31]. Whether there is a clinically relevant difference in virulence between vancomycin-resistant enterococci and vancomycin-susceptible enterococci, or between different enterococcal species is unknown.

The only variables found to be independent risk factors for mortality in our study were ICU admission and current corticosteroid therapy. ICU admission is associated with severe sepsis and shock, which are known to be risk factors for mortality in patients with BSI [7]. Patients receiving corticosteroid therapy mainly corresponded to debilitated patients with severe uncontrolled underlying disease, who are known to be a risk group for poor outcome.

Patients with E. faecium BSI in our study were more likely to receive inadequate empirical antibiotic therapy. Inadequate empirical antibiotic therapy has previously been reported to be associated with mortality, especially in patients with Gram-negative BSI and in those with vancomycin-resistant enterococcal infections [32]. However, this association was not observed in our study. A retrospective study by DiazGranados et al. identified vancomycin-resistance as a risk factor for mortality in neutropenic cancer patients. However, it was not associated with inadequate empirical antibiotic therapy, but rather was attributed to prolonged duration of BSI [15]. Factors influencing mortality among cancer patients are often difficult to asses.

The emergence of E. faecium in immunosuppressed patients with cancer is a concern, since there are limited therapeutic options for these organisms. Although new antimicrobials, such as linezolid, daptomycin and quinupristin/dalfopristin, have recently been developed to treat serious enterococcal infections, resistance to these agents has already emerged [33-35].

This study has a number of strengths. It describes the incidence of E. faecium BSI in a large cohort of BSI episodes prospectively collected in a specific immunosuppressed high-risk population. Additionally, it provides information regarding the E. faecium clonal complexes identified during the study period. However, it also has certain limitations. The small number of patients in the two groups may have prevented us from identifying significant differences between them. Also, as this was a single-centre study the results may have been influenced by local epidemiological variables, thereby limiting their applicability to other settings.

In conclusion, we found a significant increase in vancomycin-susceptible E. faecium BSI among cancer patients, especially those treated previously with carbapenems. Clonal complex 17 was responsible for the large majority of E. faecium infections, particularly in recent years. The emergence of E. faecium among immunosuppressed cancer patients is worrying since there are limited treatment options and it may presage the emergence of vancomycin-resistant enterococci. Addressing this trend for enterococci requires a rational approach that combines infection control with antimicrobial stewardship.

Supporting Information