Characterization of Enterococcus Isolates Colonizing the Intestinal Tract of Intensive Care Unit Patients Receiving Selective Digestive Decontamination

Abstract

Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5) Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU patients.

Keywords: Enterococcus; antibiotic prophylactic therapy; antibiotic resistance; intestinal colonization; selective digestive decontamination; virulence factors.

Figure 1

Overview of the dynamics of colonization by Enterococcus species and carriage of antibiotic resistance and virulence genes during and after ICU hospitalization. The black dots indicate days where fecal samples were taken during hospitalization. Discontinuation lines (//) indicate samples collected during the post-ICU period. The different species isolated are indicated by differently colored dots: orange (E. faecalis), green (E. faecium), dark gray (E.sp). Isolates not connected to a black dot were obtained from the sample closest to the left. The presence of antibiotic resistance genes is indicated by red (ermB) and purple dots (vanC1). Virulence factors are shown in heptagonal shapes (a-asa1), (e-esp-fm and esp-fs), (h-hyl). Patients that developed nosocomial infections during ICU stay with E. faecalis and E. faecium are indicated by green (E. faecium) and orange (E. faecalis) triangles. Gray boxes indicate systemic antibiotics given under clinical indications at the specific time point indicated (ERY, erythromycin; VAN, vancomycin; CPR, ciprofloxacin; CTR, ceftriaxone; CAZ, ceftazidime; CZL, cefazoline; MER, meropenem; FLX, flucoxacilin; AMX, amoxacillin; CLN, clindamycin; MTZ, metronidazole; MUP, mupirosin), yellow boxes indicate systemic antibiotics given under clinical indications during the entire ICU stay (CRX, cefuroxime; COT, cotrimoxazole).

Figure 2

16S rRNA gene-targeted qPCR based quantification of the enterococcal population present per patient in the samples taken at different time points [during ICU (D) and Post-ICU (P)].

Figure 3

Sequence types (ST) identified per sample, per patient during and after SDD therapy. The differently colored dots indicate the species: orange (E. faecalis), green (E. faecium). Numbers indicate the sequence types. Black dots indicate the time point (days) where samples were taken during hospitalization. If multiple strains were isolated and characterized, these are shown to the right of each indicated timepoint.