Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer

Abstract

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

Keywords: PBP5 mutations; PonA; ampicillin resistance; chromosomal transfer; phylogenomic diversification.

FIGURE 1

Clonality and hybridization assays with pbp5 probes of wild type, recipients and transconjugants strains. (A) PFGE SmaI digested DNA of recipient strains (1-E. faecium GE1, 25-E. faecium 64/3, 28-E. faecium BM4105RF), wild type (2-VD79C1, 4-SN71, 6-SN133, 8-HPH2, 10-H323, 12-70411, 14-H207, 16-E49, 18-E233, 20-E169, 22-E4) and transconjugants (3-GEVD79C1.5, 5-GESN71.1, 7-GESN133.1, 9-GEHPH2.1, 11-GEH323.3, 13-GE70411.2, 15-GE207.1, 17-GEE49.1, 19-GEE233.1, 21-GEE169.3, 23-GE28798.1, 24-GEE4.1, 26- 64SN71.1, 27- 64HPH2.1, 29- BMSN71.1, 30- BMH207.3, 31- BM70411.5, 32- BME169.3, 33- BMVD79C1.6). (B) Hybridization assays with a pbp5 probe (primers P1 and P2-Figure 4). M- Low Range PFGE Marker, kb (New England, BioLabs). Abbreviations: TCEfm- Transconjugant E. faecium.

FIGURE 2

Representation of the transferable chromosomal genetic platform containing pbp5. Mapping and annotation (using KEGG database) of transferable pbp5 genetic platform of transconjugant TCGEHPH2.1 (represented by black line) using E. faecium DO as reference genome. Lines above E. faecium DO genetic structure represent the transferable pbp5 genetic platform observed in wild type and transconjugant isolates (pink) and genomic regions of E. faecium GE1 recipient and transconjugant (green). GC content was calculated using seqinr in Rstudio. The window used to calculate the GC content was 200 bp, represented in the figure by each vertices of the graph. WT, wild type.

FIGURE 3

Comparison by MAUVE of the transferable genetic platform containing pbp5 (TCGEHPH2.1) with the four close E. faecium genomes (DO, Aus 0004, Aus0085 and NRRL B-2354), present in GenBank database. Each color block represents a genome region present in at least two of the sequences analyzed. These similar blocks can be identified by their color. Blank regions within the block represent point mutations or even regions that are absent in the same color blocks of the other sequences.

FIGURE 4

Characterization of E. faecium pbp5 genetic environment by PCR and sequencing. The Roman numbers I, II, III, and V represent the different pbp5 genetic platforms detected in E. faecium from this study. The numbers IV, VI to XXI were detected in available genomes from GenBank database. The different types were named according to diversity of insertions sequences, genomic fragments or conjugative transposons within genes or intergenic regions. Mutations or recombinations within genes or intergenic regions were not considered for type classification. The Table indicates the primers used (designed for this study; P1/P2 described by Dahl et al., 2000) and the size of PCR amplicons from genetic environment of types I–III and V. The A, B, C and D lines of the bottom right side figure represent RFLP patterns of amplicon P3-P2 of mobile platforms I (pattern C) and II/III (pattern D) of isolates included in this study, when digested with DdeI restriction enzyme. The patterns A and B correspond to the amplicons of the recipient strains E. faecium BM4105RF and 64/3, respectively. ^a These gene has an extra stop codon within its sequence. Abbreviations: N-acyl, (N-acyl-glucosamine-6-phosphate-2-epimerase); HP (hypothetical protein); ftsW (cell cycle protein); psr, (pbp5 synthesis repressor); pbp5 (gene encoding penicillin binding protein 5); AceT (acetiltransferase); HisPh (Histidinol Phosphate Phosphatase).

FIGURE 5

Phylogenetic analysis of PBP5 protein sequences from E. faecium isolates of this study and available in GenBank database (last update February 2014). The Maximum Likelihood tree was obtained using Mega 7 and the JTT method. The tree with the highest log likelihood (-2000,3736) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model. Then, the topology with superior log likelihood value was selected. The analysis involved 75 amino acid sequences. All positions containing gaps and missing data were eliminated of the analysis. The final dataset had a total of 445 positions. Evolutionary analyses were conducted in MEGA7. Bootstrap values are indicated and are based in 1000 permutations. The cut-off used was ≥70%. “C” followed by a number represent PBP5 amino acid combinations described in Supplementary Table S6. Only one E. faecium isolate carrying representative PBP5 sequences (each type of “C”) was included in the tree. The pbp5 genetic environments types described in Figure 4 are indicated by colored circles or by colored squares when more than one type was detected in isolates carrying the same “C.” No symbol reflects that the genetic environment was not elucidated. NA, not applicable.

FIGURE 6

Comparison of gene tree topologies (Core Genome phylogeny vs. pbp5 phylogeny). Strains of clade B are represented in blue, those of Clade A1 in red and those Clade A2 in green. The edges join the core genome and the corresponding pbp5 of each strain. Both trees were made by ML using GTR-CAT. Tress and edges were built using APE packages. The results of Mantel test (r = -0.03 and significance of 0.761) show the rearrangement of the pbp5 in comparison with the core genome. The Mantel test (APE package) was made using the distance matrix calculated from nucleotide alignments.