Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis

Abstract

Biofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism's distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.

Keywords: antimicrobial resistance; biofilm; catheter-related bloodstream infections; microscopy; polymicrobial biofilms; whole genome sequencing.

Figure 1

Relative fold-change in gene expression of C. parapsilosis. (A) Chart represents relative changes in expression of the CDR1, MDR1 and ERG11 genes in isolates of C. parapsilosis HC and CVC compared to standard C. parapsilosis CDC317 that was set up as a control and normalized to the value of 1. (B) Chart represents relative changes in expression of the CDR1, MDR1 and ERG11 genes in CDC317 strain, isolates of HC and CVC after 5 h incubation in the presence of sub-inhibitory concertation of FLU (2 µg/mL) compared to non-treated samples of corresponding isolates set to 1. A p < 0.05 (*) was considered statistically significant; p < 0.01 (**) highly significant; p < 0.0001 (****) highly extremely significant.

Figure 2

Central venous catheter colonization. Representative scanning electron micrograph of the lumen of a CVC removed from a patient with a catheter-related bloodstream infection. Rod-shaped bacteria (red arrows) and fungi (black arrow heads) colonized the CVC coexisting with host factors such as red blood cells, as can be observed with more detail in the inset. Scale bar: 10 µm.

Figure 3

Biofilm assay. Single and dual biofilms of E. cloacae complex (E. bugandensis) and C. parapsilosis were monitored using crystal violet assay. Dual biofilms were either initiated by C. parapsilosis (First C. parapsilosis) or E. cloacae complex (First E. cloacae complex) or both microorganisms (C. parapsilosis + E. cloacae complex). The dashed line (---) highlights the OD_570nm value corresponding to the cut-off value for strong biofilm producers (SBP) according to Stepanović criteria [32]. A.U.: arbitrary units.

Figure 4

Dual biofilms. Biofilm started by a mix of C. parapsilosis and E. cloacae complex (E. bugandensis) (A), C. parapsilosis (B) or E. cloacae complex (E. bugandensis) (C) on polystyrene plates. Detail of mixed biofilm, started by the two microorganisms, on polystyrene (D) and polyurethane (E) acquired by SEM. Section of a dual biofilm assembled in the lumen of a polyurethane central venous catheter (F) acquired by FIB-SEM.

Figure 5

Dual biofilms of E. cloacae complex (E. bugandensis) and C. parapsilosis assembled on glass and polyurethane. Biofilms assembled on glass were monitored using FISH with the bottom (A), intermediate (B) and top layers (C) shown with E. cloacae complex (E. bugandensis) (green) and C. parapsilosis (red). Biofilms assembled within the polyurethane CVC lumen were monitored using FIB SEM with the bottom, intermediate and top layers shown in (D,E,F), respectively.

Figure 6

Impact of host factors on dual biofilm assembly. Dual biofilm biomass assessed with crystal violet assay on control (PS) and PS conditioned for 24 h with different concentrations of human serum (HS) and plasma (HP). A p < 0.05 (*) was considered statistically significant.