Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-020-00825-3.

human isolates recovered in the same time period from Denmark, Portugal, Spain, Switzerland, and the United States [5] .The plasmid described in the previous study [5] carried Tn 1546 variant, termed 'D' [5,6] , which corresponded to deletions in orf1  and the SNP position G8234T.In our study, type A plasmids showed the same Tn 1546 variant .However, the Tn 1546 variant present in plasmid E0656_2 carried an extra SNP position C4947T .Tn 1546 is flanked by IS1216 elements upstream and downstream from the transposon (Additional File 2 : Fig. S8).This RepA megaplasmid also encodes for the erm(B) gene conferring resistance to erythromycin and a copper resistance operon tcrYAZB which has been shown to be implicated in tolerance to high levels of this heavy metal [1] .This plasmid also encodes the TA system ω-ε-ζ previously described in poultry isolates [7] .The mobilisation of this plasmid type between distinct hosts, healthy humans and pigs, could be explained by the presence of the type IV secretion gene traG .This traG gene has been previously reported in pIP501, a conjugative broad-host-range plasmid present in nosocomial E. faecium and E. faecalis isolates [8] .
The The Tn 1546 variant present in this type is characterized by the insertion of the IS 1251 -like element (5820) and SNPs at positions T7658C, G8234T and C9692T.Two plasmids, E7246_6 and E8423_3, showed the additional SNP G4351T.We also observed deletions in orf1 , orf2 mostly between the coordinates 1-3702 but also in the coordinates 1-3343.This Tn 1546 variant was previously termed as 'C' because of the presence of the IS 1251 -like element [9] .Also in this plasmid, Tn1546 was surrounded by two IS 1216 elements ( Additional File 2 : Fig. S8).Plasmid type B also contained other AMR genes including: i) erm(B) , erythromycin resistance, ii) aph(3') , aminoglycoside resistance and iii) cfr(C) , linezolid resistance.In addition, this plasmid contains the TA axe-txe complex first described in the E. faecium pRUM plasmid [10] .
The plasmid type C was composed of seven sequences including four PLSDB complete plasmid sequences (KX810026.(1-3676) and in the intergenic region 8650-8827 or 8650-8925.As previously described, the specific coordinates of the deletions could differ between the isolates while the rest of SNP coordinates or IS-elements were identical.In this plasmid type, we also observed the presence of other AMR genes including ant( 6 .This plasmid has been previously described as pVEF4 and characterized in poultry isolates from Norway and Denmark [11] .Plasmid type E reported here, contained functional and truncated str genes conferring resistance to streptomycin.We uniquely observed two components, ω-ε, of the TA system previously described in E. faecium plasmid sequences derived from poultry isolates [7] .previously reported [12] .Notably, in the plasmid type F, the Tn 1546 element was split into blocks which were surrounded by IS 1216 elements (Additional File 2 : Fig. S8).The vanR and vanS genes were separated (> 10 kbp) from the rest of the vanA gene cluster (Additional File 2 : Fig. S8).In some plasmids E6020_3, E6988_5, E7025_5 and E7067_5, we observed other AMR genes including erm(B) , aph(3')-III , ant( 6)-Ia and lnu(B) linked to the co-integration of a Rep2-like plasmid.In all the plasmids from this type, we observed the presence of the TA system ω-ε-ζ.

B) Characterization of the predicted plasmid bins
To elucidate gene content and synteny of these plasmid bins, we integrated the nine plasmid types (A-I) derived from complete plasmid sequences (Table 1, Fig. 2) with the plasmid bins predicted by gplas (Additional File 2 : Fig. S3).We calculated Mash distances (k = 21, s = 1,000) between the complete vanA plasmid sequences (n = 26), PLSDB retrieved plasmid sequences (n = 60), and the predicted vanA plasmid bins (n = 282).The presence of edges connecting complete plasmids and predicted vanA plasmid bins revealed that the predictions had a similar k-mer content and thus further validated the predicted vanA plasmid bins (Additional File 2 : Fig. S3).Furthermore, this approach also allowed to elucidate the content and structure of the plasmids present in the predicted network.Due to the fragmented nature of the predicted plasmid bins, the Tn 1546 characterisation was performed considering uniquely SNP and deletions respect to the original transposon sequence described by Arthur et al [3] .
We focused on the presence of eight distinct groups of plasmid bins which corresponded to subgraphs highly interconnected in the network with more than 10 nodes (Additional File 2 : Fig. S3a, Table 2).
Predicted group (plasmid bins) 1 represented a novel plasmid type, termed J, which was not linked to one of the nine plasmid-types included (A-I) (Fig. 3).This was the only predicted group without a complete sequence co-clustering in the network.Isolates carrying this novel plasmid type J (n = 11) belonged to SC10 (Table 2).The majority of the isolates were from Utrecht and isolated in April-May 2012 (n = 9).However, we also found two isolates from May 2013 (E7837) and March 2014 (E8046) which were present in nearby Dutch cities (Ede -Gelderland region and Amersfoort -Utrecht region).All isolates (n = 11, 100%) shared the same Tn 1546 variant (MN) with the SNPs T7658C and G8234T.
Plasmid bins in the group 3 co-clustered with plasmid type C (n = 76) (Fig. 3).Isolates carrying this plasmid type belonged mostly to four distinct SCs: 13,12,1,10 (Table 2) which suggested horizontal spread of the plasmid type C between non-clonal isolates.Furthermore, the predicted plasmids were widely observed in the Netherlands during the entire collection period (2012-2015).Tn 1546 characterisation identified the variant MNI characterised by deletions in the orf1 , orf2 region (1-3417 in 53.1% and 1-3676 in 32.8% cases) and in the intergenic region (8650-8827).We observed an independent plasmid bin group 2 mainly formed by isolates belonging to SC13 (Table 2) but geographical widespread in the Netherlands between 2012 and 2015 (Fig. 1).Detailed analysis of the predicted plasmid types of isolates E7313, E8014, E8414 with associated complete plasmid sequences, revealed that these plasmids were linked to both plasmid bin groups 2 and 3, while their complete plasmid sequences belonged to a single plasmid type C. Furthermore, both predicted vanA plasmid bins (2, 3) shared the same Tn 1546 variants.These observations suggested that the predicted plasmid bins groups 2 and 3 should be merged and named type-C.
Plasmid bins belonging to the group 6 co-clustered with plasmid type D (Fig. 3).Isolates carrying this plasmid type mainly belonged to isolates with SC20, SC18, SC1, and SC22.They Plasmid bins in the group 7 co-clustered in the network together with plasmids type A (n = 12) (Fig. 3).Isolates carrying this plasmid type belonged to three distinct SCs : SC29, SC30, and SC32 (Table 2).All isolates were recovered from a single Dutch province (North Holland) spanning Plasmid types G and H, identified in the fully assembled strains, did not have connections to the predicted plasmid bins in the network (Fig. 3).This suggested that these plasmid types were clearly distinct to the predicted plasmid bins and were not present in the Dutch collection.
)-Ia and erm(B) conferring resistance to aminoglycoside and erythromycin, respectively.Plasmid type D is an Inc18 plasmids with a size ranging from 40.2 kbp to 47.9 kbp (mean = 43.1.5kbp, median = 41.1 kbp).The Tn 1546 variant present in this plasmid type contained an insertion of ISEf1 (9051) for the variants present in the plasmid E6055_4 and E8040_4 whereas the SNP G7747T was found in all variants.However, the coordinates of deletions present in Tn 1546 were different between the isolates bearing the plasmid type D. The Tn 1546 variant present in the plasmid E6055_4 showed a deletion in vanZ (10490-10850), the transposon variant in the plasmid E8040_4 presented deletions in vanS (5895-5932) and the intergenic region (10706-10850) whereas the variant found in E8202_3 presented a deletion between the genomic coordinates 9027-10850 .In the plasmid type D, the Tn 1546 variant showed the presence of an IS 1216 element downstream of the vanZ gene (Additional File 2 : Fig. S8).The plasmid type D also encoded the erm(B) gene conferring resistance to erythromycin.Plasmid type E included seven plasmid sequences, five from the PLSDB database (MG674582.1 , NC_008768.1,NC_008821.1,NC_010980.1,NC_011140.1)and two previously described at Arredondo-Alonso et.al 2020 (E4227_3, E4239_3).In these 2 plasmid sequences, we observed that the plasmid type E was a multireplicon Inc18 & Rep3 plasmid with a size ranging from 45 kbp to 46 kbp (mean = 45.8 kbp, median = 45.8 kbp).We observed the original Tn 1546 variant, also termed as 'A', with no deletions, IS elements or point mutations present in comparison to the sequence described by Arthur et al. [3] Plasmid type F was composed of 14 plasmid sequences including seven PLSDB complete plasmid sequences (NZ_CP013996.1,NZ_CP019210.1,NZ_CP019995.1,NZ_CP027500.1,NZ_CP041269.1,NZ_CP041279.1,NZ_LT603681.1)and seven plasmid sequences described in Arredondo-Alonso et.al 2020 (E6020_3, E6988_5, E7020_5, E7040_5, E7067_5, E7070_9, E7207_6).In these last 7 plasmid sequences, we could characterize the plasmid type F as a multireplicon Inc18 & Rep3-like plasmid, with an additional Rep2-like element in some isolates.The size of the plasmids ranged from 38.6 kbp to 63.68 kbp (mean = 48.2kbp, median = 48.2kbp).This plasmid contained a Tn 1546 variant carrying IS 1216 insertions at positions 5768 and 8839 with a deletion in orf1 (1-2547).The IS 1216 insertion in the position 8839 has been
three years (2012 to 2015).The Tn 1546 characterisation revealed that all plasmids (predicted and completed) shared a SNP position G8234T and deletion of orf1 (1-119) (6M variant).The predicted group 8 shared connections with the plasmid type termed E. However, we only observed single node in the component connected to the complete sequences co-clustering with plasmid type E (NC_008768.1, NC_008821.1,NC_011140.1)which indicates that this predicted group 8 may share some k-mer modules with the plasmid type E. All isolates (n = 10) carrying this vanA plasmid type belonged to SC18 and were isolated in Venlo (Limburg province) during September-October 2014.Tn 1546 identified in this plasmid was identical to the original transposon described by Arthur et al. [3] .