Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/ mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridiumscidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.
Enterococci are part of the regular intestinal microbiota in humans and animals. In the past two decades, E. faecium has rapidly evolved as a worldwide nosocomial pathogen by successfully adapting to the hospital environment [1,2]. The World Health Organization (WHO) has described vancomycin-resistant E. faecium (VRE) as a high-priority pathogen with urgent need of new treatments [3]. In the present study, we present a case infected with a borderline vancomycinsusceptible E. faecium #21122516 with a minimum inhibitory concentration (MIC) of 4 µg/mL, which grew on a chromogenic VRE-selective agar after a 48 h incubation but was PCR-negative for all known van genes.

Case report
In January, 2021, a woman in her 50s was admitted for cancer surgery to one hospital in Belgium. Her postoperative status in the intensive care unit (ICU) was complicated by the occurrence of ventilatorassociated pneumonia and bacteraemia for which she received multiple courses of broad-spectrum antibiotics (ceftazidime, meropenem, piperacillintazobactam, vancomycin, ciprofloxacin) spanning over a 12-week period. The patient recovered and was transferred after 3 months to a physical rehabilitation unit. Upon admission to the ICU, multiple screening swabs (rectal, upper and lower respiratory tract) taken to detect extended spectrum beta-lactamases, carbapenemase-producing Enterobacteriacae and methicillin-resistant Staphylococcus aureus were negative. However, screening for VRE was not done. After the end of antibiotic therapy (early March 2021), one routine urine specimen yielded a mixed growth of bla NDM-1 carbapenemase-producing Enterobacter cloacae and of E. faecium #21122516, which on BD Phoenix PMIC Panels (New Jersey, United States (US)) was borderline susceptible to vancomycin (MIC 4 µg/mL) and susceptible to teicoplanin (MIC < 1 µg/mL). Because this E. faecium strain also grew on a chromogenic selective agar (Chrom ID VRE, bioMérieux, Marcyl'Étoile, France) after 48 h, the isolate was sent as a putative VRE to the Belgian National Reference centre for Enterococci (UZA, Antwerpen, Belgium) for genetic confirmation.

Identification of a novel van gene cluster in a clinical Enterococcus faecium isolate not harbouring any known van genes
Genomic DNA was isolated by use of MagAttract HMW DNA kit (Qiagen, Hilden, Germany) library preparation (Nextera XT), sequenced by MiSeq (2 × 250 bp, Illumina, San Diego, US). The sequencing data of this study are deposited under the BioProject ID PRJNA741920. Data analysis was done using BacPipe v.2.6.1 [4]. BacPipe results showed the absence of van genes after screening against Resfinder and CARD databases. However, Resfams gave hits to putative glycopeptide resistance genes [5]. The hits containing scaffolds were further analysed using CLC Genomics workbench v.20.1 (clcbio, Aarhus, Denmark) and identified as follows: vanH-vanP-vanX-vanR/walR-vanSG/   In order to confirm the genetic modifications, whole genome sequencing of in vitro clones from the second and third passages showed a non-synonymous mutation (G to T) in the ligase gene (vanP) (changing Glu 335 to a stop codon) compared with the original strain and with clones from the first passage. This premature stop codon led to a ligase open reading frame 24 nt shorter than that in the parental and once passaged strains (Figure 2). Acquired vancomycin and teicoplanin resistance of E. faecium #21122516 clones was shown to be stable in vitro after five consecutive passages after the appearance of resistance in an antibiotic-free medium.

Acquisition of a large genomic region including a novel van gene cluster on a putative ICE element
We screened the van gene cluster-containing scaffold for an integrative conjugative element (ICE) using ICEberg 2.0 (http://db-mml.sjtu.edu.cn/ICEberg/) [6]. . On the other hand, we did not find any similarities to E. faecium or any enterococcal genomes when we searched against the NCBI nr database (Figure 1).

Molecular analysis of D-Ala-D-X ligases from Enterococcus faecium #21122516
In order to assess the new gene D-Ala-D-X ligase group, we did a phylogenetic analysis of the protein sequences of all known ligase genes taken from GenBank. We further divided the D-Ala-D-X ligases into six major groups based on their phylogenetic relationships. Three were D-Ala-D-Ala ligases, two were D-Ala-D-Lac and one was a D-Ala-D-Ser ligase. The new ligase gene vanP was distantly related to other D-Ala-D-Lac sequences, but clustered within the VanA group ( Figure 3).

Genetic characterisation of Enterococcus faecium #21122516
The E. faecium #21122516 strain belongs to sequence type ST1486 of the CC17 complex. In line with the characteristics of a hospital-adapted lineage, this strain harboured multiple resistance genes for trimethoprim (dfrG), tetracycline (tetO), aminoglycoside (aac(6')-Ii, ant(6')-Ia), aph (3')-III, macrolide (msr(C) and virulence factors involved in adhesion to collagen and to adhesive matrix molecules like acm (a collagen adhesin precursor), sgrA, a cell well-anchored protein that stimulates surface adhesion, ecbA collagen-binding protein A and the esp enterococcal surface protein, which promote biofilm formation. The strain harbours rep14a, rep2 and repUS15 plasmid replicons. We detected eight incomplete phages using the PHASTER online tool [7], indicating that the genome is prone to acquiring new genes.
We looked into the progenitor of E. faecium ST1486 harbouring the van gene cluster. We performed the gene-by-gene approach to determine the core genome [8] multilocus sequence typing (MLST) and whole genome MLST allelic loci distance using chewBBACA [9] and visualised it through Grapetree [10] of the strains sequenced in the past 5 years (Figure 4). A total of 74 E. faecium clinical strains from CC17 complex were randomly selected among the isolates to the reference centre in the period from 2017 to 2021 (Figure 4).

PCR screening for the new vanP gene
The new vanP gene can be detected by a standard PCR-based screening assay using primers VANP-IF 5' ATGCAGACATTCTTCCTTATCC 3' and VANP-IR 5'

Ethical statement
Approval by an ethics committee was not required for this study because the informed consent from the patient was obtained before investigation. All data were anonymised to protect patient privacy and confidentiality.

Discussion
We found a newly acquired glycopeptide resistance van gene cluster(vanP) in a clinical strain of E. faecium which had occurred as a sporadic isolate in a hospitalised patient following multiple courses of Isolates are displayed as circles. Numbers in brackets refer to the number of isolates. The distance between S1486 and ST117 was 79 allelic loci, and both sequence types belonged to the CC17 cluster.
antimicrobial therapy. Despite the fact that molecular screening for all known van genes yielded a negative result, we found that resistance to vancomycin and to teicoplanin could be acquired easily and rapidly following exposure to subinhibitory vancomycin concentrations. We could also show that a non-synonymous mutation in the ligase gene created a premature stop codon in the passaged clones, associated with an increase in the vancomycin and teicoplanin MIC. However, actual functional modifications are yet to be explored.
An increase in VRE isolates has been attributed to clonal expansion following gene acquisition [11]. We propose that ST1486 has arisen independently through horizontal gene transfer in hospitals [12]. Roseburia is a Gram-positive intestinal bacterium, and member of Clostridiales, obligate anaerobes that are motile and reported to be difficult to culture and highly susceptible to antimicrobial agents, but homologous vanGlike elements have been reported in Roseburia spp. isolates from the human intestine [13]. As for other types of enterococci, the acquisition and carriage of antibiotic resistance genes is one possible explanation for why E. faecium could spread in a hospital setting so quickly [14].
Of the 12 identified van genes, nine have been observed in clinical isolates (vanA, B, C, D, G, L, M, N) [2,15,16]. It is acknowledged that the predominance of vanA and vanB clusters in major outbreaks is partly due to these clusters being part of successful mobile genetic elements that can disseminate between successful clones. However, selection bias may also contribute to this dominance since in most studies, screening is only performed for the vanA and vanB genes [2].

Conclusion
We found a novel van gene cluster, vanP, which was associated with a low level of expression of resistance to vancomycin in a patient clinical isolate of E. faecium but caused high-level vancomycin resistance and mediumlevel teicoplanin resistance after exposure to sub-inhibitory concentrations of vancomycin. We hypothesise that this novel van gene cluster was most probably acquired during co-colonisation of Clostridium scidens and Roseburia sp. 499 via horizontal gene transfer under selective pressure by antibiotics. These data suggest that pathogenic enterococci can acquire new vancomycin resistance genes from non-pathogenic species, which can be missed when performing routine screening for vancomycin resistance that is limited to vanA and vanB resistance genes. There is a risk that if they remain unnoticed, some of these strains could proliferate in the hospital setting and pose a threat in the future.