Phylogenomic insights into evolutionary trajectories of multidrug resistant S. pneumoniae CC271 over a period of 14 years in China

Abstract

Background: Streptococcus pneumoniae is a gram-positive opportunistic pathogen, and infection risks of S. pneumoniae can be profoundly augmented by its acquired multidrug-resistance (MDR). The rapid development of MDR in S. pneumoniae was attributed to the international dissemination of a small number of multidrug-resistant "clones." Clonal complex (CC) 271 is a prevalent MDR CC in the world and the most prevalent CC in China. However, the evolutionary trajectories of multidrug-resistant S. pneumoniae CC271 in China still are largely unknown.

Methods: We investigated a collection of 1312 S. pneumoniae isolates collected from 28 tertiary hospitals in China from 2007 to 2020. Recombination prediction and recombination-masked phylogenetic analysis were combined to determine the population structure and mode of evolution of CC271. Data from the Global Pneumococcal Sequencing program (GPS) were combined to understand the global distribution of clones identified in this study. Bayesian analysis were recruited to analysis the evolutionary dynamics of dominant clones within CC271 in China.

Results: The phylogenomic analysis resulted in the discovery of two globally distributed clones, ST271-A and ST271-B. ST271-A was a derivative of ST236 and an ancestor of ST271-B and ST320, refining the internal phylogenetic relationship of CC271. ST271-B was the most dominant clone in China, with higher β-lactam resistance especially for cephalosporins comparing to other MDR clones. Bayesian skyline plot showed a rapid expansion of 19F ST271-B from 1995 to 2000, which correlates with the widespread use of cephalosporins in the 1990s in China. 19A ST320, a vaccine-escape clone, is the second largest population in China. The Bayesian skyline plot showed that the 19A ST320 began to expand rapidly around 2001, which appeared to coincide with the prevalence of 19A after application of PCV7 in 2000 in the USA. We also observed frequent transmission of 19A ST320 between countries. It suggests that mass vaccination in some countries could affect the prevalence of clones in unvaccinated countries in the context of high-frequency international transmission.

Conclusions: Our results refined the internal phylogenetic relationship of CC271, showing that the 19F ST271-B and 19A ST320 evolved independently from ST271-A, with different histories and driving forces for their evolution and dissemination in China.

Keywords: CC271; Multidrug-resistant Clone; Streptococcus pneumoniae; Vaccine escape; Whole-genome sequencing.

Fig. 1

Molecular typing results and maximum likelihood tree of 1312 S. pneumoniae isolates. The tree was constructed based on 1100 core genes and midpoint-rooted. The color strips represent the clonal complex (CC) and serotype from inside out. The main CCs and serotypes are represented by distinct colors, while other CCs and serotypes are uniformly displayed in white

Fig. 2

Phylogenetic tree and predicted recombination events of 526 CC271 isolates in China. a Recombination-masked WGS phylogeny; colors of the branches correspond to groups, which consist primarily of isolates with the corresponding ST and serotype. Red represents 19F ST236 (including Taiwan19F-14), orange represents 19F ST271-A, blue represents 19F ST271-B, purple represents 2 19F ST320 isolates, green represents 19A ST320, and gray represents a group consisting of other rare STs that are not focused. Blue and green shaded box represents the monophyletic group of 19F ST271-B and 19A ST320. b The predicted recombinations in CC271 isolates. Column corresponds to location in the reference genome, and row corresponds to taxon in the phylogeny. Red blocks represent putative recombination events shared by multiple isolates through common descent, and blue blocks represent putative recombination events occurred in a single isolate. c Annotation of the S. pneumoniae Taiwan19F-14 reference genome. Each block represents a coding sequence. Coding sequences of pbp2x, cps locus, and pbp2b are colored in red. pbp2x is located upstream of cps locus

Fig. 3

Phylogenetic tree of PBP amino acid sequences and β-lactam resistance phenotype comparison of groups in CC271. a, b Phylogeny of PBP2b (a) and PBP2x (b) amino acid sequences of 526 CC271 isolates. Bolded branches with colors are the manually selected representative sequences that correspond to each group and are identical or less different, and a few black branches that are not bolded represent PBP sequences with distinct variation that may be due to additional recombination and are not focused here. PBP2b sequence of Taiwan19F-14 (marked in figure) reference genome is similar but different from those of 19F ST236 isolates in our sample. All 19F ST271-A isolates had 100% identical PBP2b sequences with two 19F ST236 isolates in our sample. PBP2b sequences of 19F ST320 and most 19A ST320 were 100% identical. PBP2x sequences of most 19F ST236 and 19F ST271-A were 100% identical. c MICs of each β-lactam antibiotic in each group. The X-axis corresponds to each group, and the Y-axis corresponds to the log2(MIC) value. The presence of a significant difference between the two groups is marked in the figure, respectively. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001, ns, not significant

Fig. 4

Bayesian phylogenetic analysis of 19F ST271 in China. To reduce the error from various sources, we used only the genomes of 19F ST271 (n = 301) from our collection. a Time-scaled phylogeny of 301 19F ST271 genomes. Nodes of the respective tMRCA (the most recent common ancestor) of 19F ST271-A and 19F ST271-B are marked with circle and triangle, respectively. The pink strip on each node represents a 95% confidence interval derived from highest posterior density (HPD) analysis for its differentiation time, corresponding to the timeline at the bottom of the figure. Clade of 19F ST271-B (n = 289) is collapsed and represented by blue-shaded triangle. b Bayesian skyline plot of genetic diversity shows sharp expansion of 19F ST271 during the 1990s. The vertical axis shows the estimated effective population size at the corresponding time. The shaded blue areas on either side of the line represent 95% HPD confidence intervals

Fig. 5

Phylogeny of 458 19A ST320 genomes from different countries, and temporal trends of 19F ST271 and 19A ST320 in China. a Bayesian skyline plot of genetic diversity shows sharp expansion of 19A ST320 around 2001. The vertical axis shows the estimated effective population size at the corresponding time. The shaded blue areas on either side of the line represent 95% HPD confidence intervals. b The proportion of 19F ST271 and 19A ST320 in our collection every 2 years. c Reconstructed recombination-masked phylogeny 19A ST320 genome, consists of samples from this study (n = 175) and GPS (n = 283). One of the 19F ST320 isolates in this study SP65 was used as outgroup. The color strip represents the country from which the sample was isolated. Bold clades include isolates from both China and the other countries, supported by ultrafast bootstrap values of ≥ 89, with the values marked on the nodes

Fig. 6

Schematic diagram of the evolutionary history of main members of CC271. Clones were represented by circles. Each junction represents a step of differentiation. The annotations above each circle show the characteristics of each clone. Annotations at each junction show changes in pbp2x and pbp2b genes or cps locus that occur during the process of differentiation. *Two global distributed clones of 19F ST271 distinguished in this study