Sequence type 17 is a predictor of subsequent bacteremia in vancomycin-resistant Enterococcus faecium-colonized patients: a retrospective cohort study

This study involved VREF-colonized patients from March 2014 to February 2015 at the Samsung Medical Center, Seoul, Korea, a large tertiary referral hospital at which more than 70% of patients were referred from other regions across the country. VREF rectal screenings were performed according to infection prevention policy and protocols of the hospital. Patients transferred to the hospital from other medical or long-term care facilities and patients transferred from a general ward to intensive care units (ICUs) were subjected to rectal VRE screening.

Two cohorts of patients carrying VREF were enrolled: those colonized with ST17 VREF and those with non-ST17 VREF. Exclusion criteria were as follows: 1) patients age under 18, 2) patients having VREF bacteremia preceding or on the same day of detection of rectal VREF colonization, and 3) Patients who died on the day of detection of rectal colonization. Subsequent VREF bacteremia events within one year of detection of colonization were recorded. All patients were observed for one year after colonization or observed until death or loss to follow-up. Clinically significant VREF bacteremia was defined as either the isolation of VREF from two or more separate blood samples, or the isolation of VREF from a single blood sample in patients with clinical symptoms and a concomitant infection [18]. Overall 30-day and 1-year survival rates were compared between VREF bacteremia group and the group of patients who did not develop VREF bacteremia.

Clinical information was reviewed during the 1-year follow-up period using electronic medical records, and data were collected on age; sex; body mass index; underlying diseases such as decompensated liver cirrhosis, severe acute kidney injury requiring renal-replacement therapy, diabetes mellitus, solid cancer, and hematologic malignancy; history of liver transplantation; neutropenia, Charlson comorbidity index [19], ICU stays; presence of central venous catheter; intubation; intra-abdominal surgery; parenteral nutrition; and specific antibiotic use prior to the onset of subsequent VREF bacteremia. The study was approved by the Institutional Review Board of the Samsung Medical Center.

Genomic DNA of the isolates was extracted using a G-spin Genomic DNA extraction kit (iNtRON, Korea) according to the manufacturer’s instructions. Multilocus sequence typing (MLST) of seven selected housekeeping loci (adk, atpA, ddl, gdh, gyd, purK, and pstS) with polymerase chain reaction (PCR) amplification was used for genotyping of VREF isolates [20]. The e-BURST algorithm was used to analyze the relatedness of each VREF isolate ST [21]. The presence of virulence genes esp and hyl was detected by PCR [22, 23], and confirmed by sequencing [24].

In cases in which both blood and rectal VREF isolates showed an identical ST, pulsed field gel electrophoresis (PFGE) was conducted to determine the clonal association. For PFGE, bacterial DNA was digested with the Sma I restriction enzyme (TaKaRa Bio Inc., Shiga, Japan) and separated by electrophoresis using a CHEF DR II system (Bio-Rad Laboratories, Hercules, CA, USA). The PFGE patterns were analyzed using Gel Compar II software (Applied Maths, Kortrijk, Belgium). Potential clonal relatedness was determined at a ≥ 80% level of similarity [25].

All statistical analyses were performed in SPSS 23.0 for Window (IBM Corp., Armonk, NY, 2015) and Stata 15.1 (StataCorp., College Station, TX, USA). Power analysis for two-sample comparison of survival analysis was implemented with fixed sample size and hazard difference [26]. A Student’s t-test or Mann–Whitney U test was used to compare continuous variables and a chi-square test or Fisher’s exact test was used to compare categorical variables. Time to development of subsequent bacteremia during the 1-year follow-up period was calculated using the Kaplan–Meier method and cohort groups were compared using a log-rank test.

Two statistical models were used to adjust the covariates to determine the association between ST17 and the development of subsequent bacteremia. In model 1, each variate was compared between ST17 and non-ST17 cohorts. Variables with a P value < 0.15 in the univariate analysis were then included in a multivariate Cox regression model to calculate an adjusted hazard ratio (aHR) for subsequent bacteremia. Model 2 analysis evaluated other risk factors for developing VREF bacteremia. After all variates were analyzed using univariate Cox regression to calculate the hazard ratio for subsequent bacteremia, variables with a statistical significance in univariate analysis and with a probable clinical meaning were selected in multivariable Cox regression. All P values were two-tailed, and values < 0.05 were considered statistically significant.

Among 254 VREF carriers identified during the study period, 221 were enrolled in the study (Additional file 1: Fig. S1). All isolates except one (99.5%) belonged to CC17. The most frequent ST was ST17 (23.5%). Among the isolates in the non-ST17 cohort, ST230 (15.8%) was the most frequent, and followed by ST981 (7.7%), ST78 (5.9%), ST192 (5.4%), ST927 (4.1%), and ST789 (3.6%) (Additional file 1: Table S1). The isolates of 98.1% of the ST17 cohort had both the esp and hyl genes, whereas 75.7% of the non-ST17 cohort had both genes (Table 1). Among underlying conditions of patients, liver transplantation was more closely associated with non-ST17.

The incidence rate of subsequent VREF bacteremia was 0.447 cases per 1000 patient-days (16 cases per 35,816 observation days). The median observation day of patients who developed VREF bacteremia (VRE-B group) was 28 (range, 3–187 days). The most frequent underlying disease in the VRE-B group was hematologic malignancy (10 of 16, 62.5%) followed by liver transplantation (3 of 16, 18.8%) (Table 2). Overall 30-day and 1-year survival rates in the VRE-B group and the group of patients who did not develop VREF bacteremia were 75.0% versus 79.9% (P = 0.615) and 37.5% versus 57.5% (P = 0.073), respectively.

The ST17 and non-ST17 cohorts included 52 cases and 169 cases, respectively. The non-ST17 cohort included 32 isolates with single-locus variants and 77 double-locus variants of ST17, among which ST230 was most frequent (Additional file 1: Table S1 and Fig. S2).

Subsequent VREF bacteremia developed in 11.5% and 5.9% of ST17 and non-ST17 cohorts, respectively (P = 0.257). Adjusted analyses using two models showed a significant association between ST17 and subsequent VREF bacteremia. The first model analysis in which virulence factors, hematologic malignancy, liver transplantation, and previous glycopeptide use were included in multivariate analysis revealed that ST17 was significantly associated with developing subsequent VREF bacteremia (aHR, 4.02; 95% confidence interval [CI], 1.32–12.29, P = 0.015) (Fig. 1). The statistical power was > 0.999, which was calculated from sample size (n = 221), ST17/non-ST17 ratio 0.38 and the aHR 4.02. In the second model, in which VREF ST17, renal replacement therapy, hematologic malignancy, liver transplantation, and previous use of glycopeptide, and carbapenem were included in multivariable analysis, ST17 showed a significant association with development of subsequent VREF bacteremia (aHR, 7.14; 95% CI, 1.83–27.83; P = 0.005). In addition, liver transplantation (aHR, 40.08 32.65; 95% CI, 5.01–329.76 4.27–249.75; P = 0.001), and hematologic malignancy (aHR, 20.97; 95% CI, 5.01–87.82; P < 0.001) were also determined to be significant risk factors for subsequent bacteremia (Table 3).

Of the 16 patients who developed subsequent VREF bacteremia, 12 VREF blood isolates were available for microbiological analysis. The ST distribution of blood VREF isolates was different from that of rectal isolates (Fig. 2). ST17 was the most common genotype (58.3%) among blood VREF isolates. Comparison of STs between rectal and blood VREF isolates showed that only 7 of 12 cases (58.3%) had identical STs. Among 7 cases with identical STs between rectal and blood isolates, 4 belonged to ST17. In the ST17 VREF carriers who developed subsequent bacteremia, all cases were caused by ST17, but in the non-ST17 carriers, subsequent bacteremia was often caused by another ST, including ST17 (Fig. 2). Among 7 VREF pairs with identical STs between rectal and blood isolates, 6 pairs showed identical PFGE patterns and 1 pair showed a 88.9% similarity (Additional file 1: Fig. S3).