The importance of adjusting for enterococcus species when assessing the burden of vancomycin resistance: a cohort study including over 1000 cases of enterococcal bloodstream infections

Enterococcus spp. are part of the normal gastrointestinal flora. Among those pathogens, resistance to antimicrobial substances, notably to vancomycin, results in limited therapeutic options [1, 2]. In recent years, hospital-acquired infections (HAI) caused by vancomycin-resistant enterococci (VRE) have emerged as a relevant burden on patients and healthcare systems globally [3–5]. In order to reduce the spread of resistant strains in hospitals, infection control measures, e.g. contact precautions, have been proposed [6, 7]. To assess the efficiency of VRE prevention measures, the mortality- and financial burden of VRE infections has to be assessed. However, the methodological approach on assessing VRE-burden remains controversial [2, 8, 9] and only few studies have addressed economic aspects [10–15]. As costs are often not available as infection-attributable costs (costs after onset for infection) length of stay (LOS) after onset of infection is being used as a surrogate parameter [2, 8, 16].

Although analyses should compare VRE infections to VSE-infected patients when the attributable effect of vancomycin resistance is addressed, [2, 8, 16] prior studies also utilized comparisons to cohorts with non-enterococcus infections [17, 18] or cohorts without infection [8, 12, 19–24]. Since the course of enterococcal infections may also be influenced by the enterococcus subspecies itself [2, 8, 9, 25, 26], analyses not considering the pathogen species may therefore be biased as result of the different virulence of the pathogens.

In a large cohort of cases with bloodstream infection (BSI), we therefore studied the influence of vancomycin resistance and enterococcus subspecies on in-hospital mortality, hospital costs and length of hospital stay.

We initially extracted n = 24,086 clinical isolates diagnosed with Enterococcus faecium or Enterococcus faecalis from the microbiology database. After excluding all isolates not derived from blood cultures and correcting for copy strains, 1242 patients with BSI caused by E. faecium or E. faecalis were included in the analysis (Fig. 1). Sufficient data on all relevant parameters was available for 96.4% of the patients. Overall, this accounted for 1160 patients, 91% with infections caused by VSE and 9% by VRE. Table 1 gives an overview of the parameters for all patients and shows the results of the (i) univariate comparison of VSEm vs. VSEf and (ii) the comparison of VSEm vs. VREm BSI cases. The highest in-hospital mortality rate was found among VREm cases (50.5%) followed by VSEm cases (39.6%) and VSEf cases (24.4%). Also regarding LOS, highest numbers were found among VREm cases (total LOS, 54 days) followed by VSEm (42 days) and VSEf (32 days). In all three groups, the Charlson comorbidity score was similar with a median of 7.

Parameter	(A) VS-E. faecium (n = 493)	(B) VS-E. faecalis (n = 563)	P-Value for A) vs. B)		(C) VR-E. faecium (n = 103)	P-Value for C) vs. A)
			P-Value	OR (CI 95)		P-Value	OR (CI 95)
In-hospital mortality	39.6% (195)	24.4% (132)	0.000	2.137 (1.638-2.787)	50.5% (52)	0.041	1.558 (1.017–2.387)
Male	58.6% (289)	63.1% (355)	0.141	0.830 (0.648-1.063)	66.0% (68)	0.163	1.371 (0.879–2.141)
Age in years, median (IQR)	67 (54–75)	64 (53–73)	0.059	n.a.	61 (52–70)	0.107
Cardiac disease	28.2% (139)	29.0% (163)	0.786	0.964 (0.737-1.259)	26.2% (27)	0.683	0.905 (0.559-1.464)
Vascular disease	22.1% (109)	22.4% (126)	0.916	0.984 (0.736-1.317)	22.3% (23)	0.961	1.013 (0.608-1.687)
Pulmonary disease	20.1% (99)	23.1% (130)	0.236	0.837 (0.623-1.124)	21.4% (22)	0.769	1.081 (0.643-1.818)
Rheumatic disease	3.0% (15)	3.6% (20)	0.644	0.852 (0.431-1.683)	2.9% (3)	0.944	0.956 (0.272-3.364)
Gastrointestinal disease	5.5% (27)	3.7% (21)	0.174	1.495 (0.834-2.680)	5.8% (6)	0.888	1.068 (0.429-2.656)
Diabetes	22.9% (113)	32.0% (180)	0.001	0,633 (0,481-0,833)	19.4% (20)	0.437	0,810 (0.476-1.379)
Renal disease	59.0% (291)	52.8% (297)	0.041	1.290 (1.011-1.647)	70.9% (73)	0.025	1.689 (1.065-2.679)
Liver disease	29.6% (146)	19.9% (112)	0.000	1.694 (1.276-2.249)	42.7% (44)	0.009	1.772 (1.146-2,74)
Cancer/ immunological disease	48.1% (237)	36.1% (203)	0.000	1.642 (1.283-2.101)	53.4% (55)	0.325	1.238 (0.809-1.894)
Neurological disease	7.3% (36)	13.0% (73)	0.003	0,529 (0,348-0,804)	8.7% (9)	0.616	1.215 (0.566-2.608)
Charlson comorbidity index, median (IQR)	7 (4–9)	7 (4–9)	0.907	n.a.	7 (5–9)	0.377	n.a.
Length of stay as Median (IQR)
LOS total (days)	42 (23–78)	32 (16–61)	0.000	n.a.	54 (36–85)	0.010	n.a.
LOS before BSI (days)	19 (9–34)	10 (2–25)	0.000	n.a.	27 (15–39)	0.003	n.a.
LOS after BSI (days)	18 (8–41)	16 (8–33)	0.308	n.a.	23 (8–45)	0.183	n.a.
LOS normal ward	15 (1–36)	11 (1–25)	0.010	n.a.	17 (0–44)	0.368	n.a.
LOS ICU	18 (1–49)	10 (1–40)	0.016	n.a.	24 (5–57)	0.050	n.a.
Hospital costs as median (IQR)
Total hospital costs	51,365 (22,535-119,789)	31,122 (11,829-74,344)	0.000	n.a.	80,465 (47,887–157,447)	0.000	n.a.
Daily costs	1,237 (729–1,812)	1,014 (614–1,466)	0.000	n.a.	1,484 (1,095–2,186)	0.000	n.a.
Medical staff	7,600 (3169-16,468)	5,344 (1841-12,213)	0.000	n.a.	10,390 (6,364–20,983)	0.002	n.a.
Nursing staff	11,499 (4,476-26,237)	7,141 (2,610-21,591)	0.000	n.a.	16,661 (8,029–32,905)	0.003	n.a.
Assistant medical technicians	2,630 (1,028-5694)	1,906 (646–4,325)	0.000	n.a.	3,665 (1,397–6,795)	0.082	n.a.
Pharmacy	6,924 (2,200-20,922)	2,742 (636–7,865)	0.000	n.a.	17,145 (8,087–36,779)	0.000	n.a.
Expenses for implants/transplants	0 (0–412)	0 (0–170)	0.005	n.a.	20 (0–605)	0.767	n.a.
Medical supply	6,858 (2,820-15,057)	3,926 (− 1,338-9,782)	0.000	n.a.	9,878 (5,104–18,957)	0.002	n.a.
Medical infrastructure	1,893 (939–4,128)	1,473 (547–3,271)	0.000	n.a.	2,629 (1,611–5,219)	0.001	n.a.
Non-medical infrastructure	8,975 (4,215-18,206)	6,176 (2,576-14,049)	0.000	n.a.	12,244 (6,763–20,002)	0.003	n.a.

Categorical variables displayed as percentage and number; continuous variable displayed as median and interquartile range. *P-value, categorical variables tested with Chi-square test, continuous variable tested with Wilcoxon rank sum test. BSI = blood stream infection, IQR = interquartile range, OR = odds ratio, CI95 = 95% confidence interval. VRE = vancomycin-resistant enterococcus, VSE = vancomycin-susceptible enterococcus. N.a. = not applicable

Bold entries represent statistically significant factors

In multivariable analyses on LOS after BSI onset among vancomycin-susceptible enterococci cases, some chronic diseases and E. faecium statistically significant increased LOS as compared to E. faecalis (see Table 2). Vancomycin-resistance was not found to additionally increase LOS among E. faecium cases (see Table 2 and Fig. 2). Regarding in-hospital death, patients with E. faecium-BSI had a higher chance for death as compared to E. faecalis-cases when only vancomycin-susceptible cases were considered (see Table 3 and 4). Among E. faecium cases, vancomycin-resistant was not found to be an additional risk factor for death (Table 4).

Parameter		VS-E. faecium vs. VS-E. faecalis					VS-E. faecium vs. VR-E. faecium
		ME	Sig.	CI 95 (lower-upper)			ME	Sig.	CI 95 (lower-upper)
Renal Disease		1.588	0000	1.891	–	3.868	1516	0.001	1.472	–	4.201
Age (years)		0.659	0000	0.969	–	0.987	Not significant
Lung Disease		1.457	0000	1.800	–	4.342	1333	0.018	1.151	–	4.409
Gastrointestinal Disease		1.323	0001	1.959	–	11.025	Not significant
Liver Disease		1.270	0.003	1.256	–	3.100	1370	0.009	1.226	–	4.155
Vascular Disease		1.216	0.016	1.104	–	2.622	Not significant
Enterococcus species	E. faecium	1.258	0.004	1.170	–	2.324	Not applicable
	E. faecalis	Reference = 1

BSI = bloodstream infection, CI95 = 95% confidence interval, ME multiplicative effect

Parameter	Discharge alive (n = 781)	In-hospital death (n = 379)	*P-value	OR (CI 95)
Vancomycin resistance	6.7% (52)	13.7% (52)	< 0.001	2.273 (1.512-3.417)
E. faecium	44.7% (349)	65.2% (247)	< 0.001	2.311 (1.792-2.979)
E. faecalis	55.3% (432)	34.8% (132)		1 = Reference
Male	61.2% (478)	62.0% (235)	0.792	1.037 (0.805-1.334)
Age (years), median (IQR)	64.0 (51–73)	66.0 (56–74)	0.011	n.a.
Heart disease	25.1% (196)	35.4% (134)	< 0.001	1.641 (1.258-2.14)
Vascular disease	19.6% (153)	27.7% (105)	0.002	1,57 (1,18-2.091)
Lung disease	19.6% (153)	26.1% (99)	0.011	1.461 (1.093-1.952)
Rheumatic disease	2.6% (20)	4.7% (18)	0.050	1.895 (0,99-3.626)
Gastrointestinal disease	4.1% (32)	5.8% (22)	0.195	1,44 (0,825-2.515)
Diabetes	26.8% (209)	27.4% (104)	0.807	1.033 (0,784-1.361)
Renal disease	46.9% (366)	78.1% (296)	< 0.001	4.055 (3.061-5.371)
Liver disease	18.2% (142)	42.2% (160)	< 0.001	3.283 (2.498-4.314)
Cancer/Immunological disease	41.4% (323)	45.4% (172)	0.194	1.176 (0,918-1.506)
Neurological disease	10.8% (84)	9.0% (34)	0.346	0,817 (0.537-1.241)

Categorical variables displayed as percentage and number; continuous variable displayed as median and interquartile range. *P-value, categorical variables tested with Chi-square test, continuous variable tested with Wilcoxon rank sum test. BSI = blood stream infection, IQR = interquartile range, OR = odds ratio, CI95 = 95% confidence interval. VRE = vancomycin-resistant enterococcus, VSE = vancomycin-susceptible enterococcus. N.a. = not applicable

Bold entries represent statistically significant factors

Parameter		VS-E. faecium vs. VS-E. faecalis					VS-E. faecium vs. VR-E. faecium
		OR	P-value	CI95 (lower-upper)			OR	P-value	CI95 (lower-upper)
Age		1015	0,002	1005	–	1025	1016	0,010	1004	–	1029
Vascular disease		1407	0,042	1012	–	1956	Not significant
Renal disease		3120	0,000	2274	–	4280	4005	0,000	2708	–	5922
Liver disease		2909	0,000	2109	–	4011	2390	0,000	1618	–	3529
Enterococcus species	VS-E. faecium	2023	0,000	1519	–	2695	Not applicable
	VS-E. faecalis	Reference = 1					Not applicable
Vancomycin-resistant		Not applicable					1.283	0.300	0.801	–	2.057
Vancomycin-susceptible		Not applicable					Reference = 1

OR = odds ratio, CI95 = 95% confidence interval

Regarding economic aspects, almost all hospital costs were significantly higher in the VREm BSI cohort compared to the VSEm BSI and the VSEf cohort.

During the last 15 years (since 2003), 4 meta-analyses on mortality- and financial burden of VRE infections [19, 22–24], and few recent studies on costs associated with VRE infections (not included in the meta-analyses) were published [13, 15, 32]. The studies demonstrate that vancomycin resistance is associated with overall worsened outcome (mortality, length of stay and hospital costs). However, although former studies indicated that E. faecium isolates might be more virulent than E. faecalis isolates irrespective of vancomycin resistance [2, 8, 9, 26], many of the above mentioned studies did not adjust for enterococcal subspecies. As Kaye et al. showed in 2004, this could lead to an overestimation of the outcome effects attributable to vancomycin resistance [9]. Some of the authors discussed this issue in their articles, arguing that meta-analyses cannot improve the quality of data published [22, 24].

In our analyses, in-hospital mortality and length of stay after BSI onset were independently associated with underlying diseases, age and E. faecium but not with vancomycin resistance.

Regarding mortality vancomycin resistance was associated with increased in-hospital mortality in the univariate analysis. This result is in agreement with recent studies including three meta-analyses [19, 22, 24]. However, after adjusting for underlying diseases, age, and species (E. faecium vs. E. faecalis), in-hospital mortality was no longer associated with VRE. There are several possible explanations for these differences with other studies.

Systemic enterococci infections mainly occur in patients with severe underlying diseases and comorbidities [33, 34] which also applies to our study cohort. Patients had a very high Charlson comorbidity score with a median of 7. In this regard, there was no significant difference between the cases infected with E. faecalis, E. faecium or vancomycin-resistant strains. Furthermore, infections caused by antimicrobial-resistant bacteria are often associated with increased morbidity and mortality [19]. These differences are explained by the delay in or even complete lack of an effective antibiotic treatment despite the availability of effective drugs [1, 24]. Prematunge et al. adjusted for appropriate antimicrobial therapy but found that the differences remained [24]. Outcome differences resulting from varying pathogenicity of enterococci species is an alternative explanation [35, 36]. In the multivariable analysis we found only small differences for in-hospital mortality resulting from vancomycin resistance, differences which are insufficient to explain the univariate results. However, we observed significantly increased mortality associated with vancomycin-susceptible E. faecium compared to vancomycin-susceptible E. faecalis.

Possibly because E. faecium is the most common type of VRE worldwide and most E. faecalis isolates from infections are vancomycin-susceptible, it might be a limitation to most existing studies that this confounder is not considered [10–16, 18–24, 32]. Prematunge et al. already pointed out in their 2016 meta-analysis, that it is possible that many observations on VRE-associated outcome are based on differences in the infection-causing species rather than on vancomycin resistance [24].

Attempts to assess the pathogenicity of enterococci go far back in time. In some of the previous studies, the pathogenic potential of commensal E. faecium was determined, whereas most of nowadays HAIs are caused by isolates of a different E. faecium lineage [37, 38]. These so-called hospital-associated strain types (formerly known as clonal complex CC17) differ from commensal human and animal isolates by a distinct core and accessory genome content. Ampicillin resistance is a phenotypic marker of these hospital-associated strain types [39–41]. It has been shown that AMP-R E. faecium isolates causing healthcare-associated infections are in fact more pathogenic than commensal variants [42, 43]. In a supplementary analysis (Additional file 1: Tables S1 and S2) we found that 95% of our E. faecium isolates were ampicillin-resistant (AMP-R), in contrast to only 1% of the E. faecalis isolates. Due to the uneven distribution of ampicillin resistance over the two pathogens we were not able to assess potential virulence differences between commensal and hospital-associated isolates. However, our results are supportive to previous population-based and molecular analyses of E. faecium from hospital-acquired infections.

Several reports have linked VRE infections with increased costs [13, 15, 19, 32]. In the univariate analyses, our data showed similar results as almost all costs were significantly higher in VREm-BSI patients than in patients with VSEm-BSI. Butler et al. reported that pharmacy costs are the second most relevant driver of increased costs, making up to 18% of the total amount [21]. In our cohort, the percentage of pharmaceutical costs for patients with VREm made up 21% of the total hospital costs while they made up only 9% of the costs of patents with BSI caused by VSEm (< 0,001). As we were not able to attribute costs to particular agents, these differences could be due to higher antibiotics costs or to differences in underlying conditions treated. For this reason, we analyzed the length of stay before and after onset of BSI. Our data showed increased lengths of stay overall and before onset of VREm-BSI, but not after. Interestingly, all BSIs were classified as HAIs as none occurred prior to day 3 after hospital admission. On average, the BSI episodes occurred on day 16 of hospitalization. Moreover, the same phenomenon was observed in the analysis of the infection-attributable LOS stratified by the two clinically relevant Enterococcus species. Cases with vancomycin susceptible E. faecium BSI were in-hospital longer before onset of infection than vancomycin susceptible E. faecalis cases. No difference in LOS was observed after onset of infection.

This study has several limitations. The cases were identified retrospectively through a microbiological database and 6.6% lacked sufficient data for this analysis. We did not have data on the course or severity of infection and the antibiotic treatment performed. We did not have separate data on true costs before and after the bloodstream infection. We did not perform molecular analyses on the enterococci isolates to assess their potential virulence traits.

In our study, vancomycin resistance in patients with Enterococcus faecium bloodstream infection was associated with increased total costs, length of stay before onset of infection, but not with infection-attributable LOS or in-hospital mortality. We observed that vancomycin-susceptible E. faecium infections were more strongly associated with increased LOS and mortality than vancomycin susceptible E. faecalis infections and might indicate higher virulence of E. faecium as compared to E. faecalis. In order to avoid overestimation of VRE-attributable effects, in addition to vancomycin-resistance species should be taken into consideration in future studies assessing the burden of VRE infections.