Virulence Factors of Streptococcus pneumoniae. Comparison between African and French Invasive Isolates and Implication for Future Vaccines

Abstract

Background: Many surface proteins thought to promote Streptocococcus pneumoniae virulence have recently been discovered and are currently being considered as future vaccine targets. We assessed the prevalence of 16 virulence genes among 435 S. pneumoniae invasive isolates from France and the "African meningitis belt" region, with particular focus on serotype 1 (Sp1), to compare their geographical distribution, assess their association with site of infection and evaluate their potential interest as new vaccine candidates.

Methods: Detection by PCR of pspA (+families), pspC (+pspC.4), pavA, lytA, phtA,B,D,E, nanA,B,C, rrgA (Pilus-1), sipA (Pilus-2), pcpA and psrp was performed on all isolates, as well as antibiotic resistance testing and MLVA typing (+MLST on 54 representative strains). Determination of ply alleles was performed by sequencing (Sp1 isolates).

Results: MLVA and virulence genes profiles segregated Sp1 isolates into 2 groups that followed continent distribution. The ply allele 5 and most of the genes that were variable (nanC, Pilus-2, psrp, pcpA, phtD) were present in the French Sp1 isolates (PMEN clone Sweden(1)-28, ST306) but absent from the African ones. Whereas all African Sp1 isolates clustered into a single MLST CC (CC217), MLVA distinguished two CCs that followed temporal evolution. Pilus-2 and psrp were more prevalent in bacteraemic pneumonia yielded isolates and phtB in meningitis-related isolates. Considering vaccine candidates, phtD was less prevalent than anticipated (50%) and pcpA varied importantly between France and Africa (98% versus 34%). Pilus-1 was carried by 7-11% of isolates and associated with β-lactams resistance.

Conclusions: Most virulence genes were carried by the European ST306 clone but were lacking on Sp1 isolates circulating in the African meningitis belt, where a more serious pattern of infection is observed. While virulence proteins are now considered as vaccine targets, the geographical differences in their prevalence could affect the efficacy expected from future vaccines.

Fig 1. Minimum spanning tree of Sp1 invasive isolates according to MLVA genotypes and either continent of origin (1a, N = 136) or ply allele (1b, N = 62).

The size of the circle reflects the number of isolates identified, the distance between circles reflects the degree of genetic divergence, the circle colors represent either the continent (1.a) or the ply allele type (1.b), the colours outside of circles indicate major clonal groupings (MLVA CCs). MLVA = Multi locus variable number tandem repeats analysis, CC = Clonal Complex, N = number.

Fig 2. Distribution of Sp1 isolates (N = 136) according to virulence genes profile by (A)) unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on similarity matrix (B) Multi-Dimensional Scaling (MDS) based on similarity matrix.

2.A: Every row represents one strain, the dark plot means the presence of the gene, colour represents the country of isolation (blue = France, red = Africa).2.B: MDS is a visualization method displaying in 3-dimensional space the information contained in the similarity matrix. Every dot represents a group of strains sharing an identical combination of virulence genes. The more different the gene combinations are, the more distant the dots are. Colours represent the region of isolation (blue = France, red = Africa).

Fig 3. Prevalence of vaccine candidate antigens among invasive isolates according to PCV13 (VTs) or non-PCV13 (NVTs) serotypes inside the French (3.a) and African (3.b) collections.

It illustrates the theoretical coverage achievable by each antigen in a monovalent or combined (PhtD+PcpA) hypothetical vaccination in terms of percentage of IPD strains circulating at the time of the study likely to express the vaccine-included protein. The ply gene is not represented since present by definition in 100% of our collection. The prevalence of the combination phtD + pcpA genes was arbitrarily calculated taking into account the presence of at least one of the two genes. Abbreviations: Sp = serotype, IPD = invasive pneumococcal disease, N = number, PCV13 = 13 valent pneumococcal conjugate vaccine, VT = PCV13 included serotype, NVT = non-PCV13 included serotype.