Among human pathogens, Streptococcus pneumoniae holds an especially prominent place in the history of biomedical investigation.  Griffith (1928) described the transforming principle, a soluble substance released by dead, virulent pneumococci that could render living avirulent pneumococci able to effectively kill a mouse.  Oswald Avery’s commitment to curing pneumococcal pneumonia (http://profiles.nlm.nih.gov/ps/retrieve/Narrative/CC/p-nid/37) led him and his collaborators to determine that the pneumococcal transforming principle was DNA (Avery et al., 1944).  It was also Avery’s and his collaborators’ work on pneumococci that provided some of the first insights into the chemical nature of most bacterial capsules.

A recent study in Science (Croucher et al., 2011) by a large collaborative group of investigators now offers another significant advance relating to both the pneumococcus and DNA.  Croucher et al. addressed key issues in pathogen evolution by applying whole-genome sequencing to 240 isolates from diverse geographic locations (on four continents) of a particular multi-drug-resistant pneumococcal strain known as pneumococcal molecular epidemiology network 1 (PMEN1) of capsular serotype 23F.  Their results offer insights into: 1) the relative importance of different sources of sequence diversification, such as horizontal gene transfer followed by recombination versus de novo mutation, and 2) the rate of evolutionary change for a clinically important bacterial pathogen.

Since the study focused on a strain with resistance to multiple antibiotics, it had to have emerged as such in the relatively recent (antibiotic-using) past.  By comparing genomic sequences of numerous isolates derived from such a common ancestor, the authors were better able to determine which regions of the genome had undergone mutation or recombinational replacement.  These circumstances permitted construction of a plausible phylogenetic tree for the isolates being studied.

The poor correlation between the dates of isolation of different strains and the corresponding phyogenetic distances from the root of the tree suggested that much of the variation was from horizontal gene transfer with a reciprocally smaller contribution from routine spontaneous base substitutions. The sites of putatively imported DNA were identified as such by virtue of the high number of polymorphic sites in proximity to one another.  Impressively, almost three-quarters of the pneumococcal genome were affected by recombination in at least one isolate, and an average of over 74,000 base pairs was replaced by each such event.  Previous studies had similarly suggested that genetic changes in pneumococci were dominated by horizontal gene transfer and recombination (Coffey et al., 1998; Feil et al., 2000).

Susceptibility to recombinational replacement of gene sequences was distributed unevenly throughout the genome.   Of clinical relevance, a number of loci located in apparent recombination hotspots encode either surface protein antigens (such as PspA, PspC, and PsrP) thought to be targets of protective antibodies or the capsule biosynthesis locus.  The capsular polysaccharide has long been (going back to Avery and his contemporaries) regarded as the chief target of protective antibody for pneumococci.  The implication is that the human immune response is exerting strong selection on the pneumococci.

A total of ten serotype switches were identified among the 240 characterized isolates.  In some instances, the newly expressed serotype was not represented in the initial seven-valent pneumococcal conjugate vaccine.  The inferred times of origin for these antigenically-modified strains suggested that, in some cases, the serotypic changes pre-dated vaccine introduction but only became prevalent due to vaccine-related selection that severely depleted existing susceptible strains thereby permitting already serotype-switched strains to proliferate.

The analysis also revealed the effects of selection for antibiotic resistance, although different classes of antibiotics appeared to have exerted selective pressures of substantially varying potency.  While aminoglycosides and chloramphenicol exerted very weak selection on this pneumoccocal clade, the macrolide antibiotics have exerted much stronger selection that has resulted in multiple independent acquisitions of resistance.

The authors are appropriately cautious in drawing conclusions.  They note that their analysis applies to one clade of one pathogen.  They cite the lack of comparable spread by another serotype 23F strain (BM4200) exhibiting resistance to multiple antibiotics.  In addition, they note that pneumococcal strains lacking substantial drug-resistance mechanisms nevertheless persist in the human population.  While the present results are valuable and impressive, it is clear that we have a long way to go before we will be able to fully assess the quantitative roles of different evolutionary mechanisms in driving pathogen diversification or be able to predict pathways of pathogen evolution with clinically-useful degrees of precision.

Nevertheless, the medical implications of the insights into pneumococcal evolution described by Croucher et al. are substantial.  Given the ease with which pneumococci can replace one capsular polysaccharide, and serotype, with another, no vaccine that elicits immunity primarily through the induction of capsule-specific antibodies and that covers only a small subset of serotypes is guaranteed to remain highly effective in the long-term.  The possibility of serotype replacement (Lipsitch, 1997,1999) must be taken seriously. Furthermore, even a vaccine based on a pneumococcal surface protein that in principle could elicit immunity irrespective of capsule-determined serotype is not likely to be “immune” from the same sort of subversion via horizontal gene transfer and recombination that applies to vaccines focused on the elicitation of antibodies to capsular polysaccharides.  Consequently, a universal pneumococcal vaccine based on pathogen-derived protein antigens and generating immunity to all capsular serotypes may well need to include a cocktail of distinct immunogens that are encoded at loci distributed widely in the pneumococcal genome and among which variants at each locus are represented.  Such compositional heterogeneity in the vaccine may be the price exacted by the protean pneumococcus for mininimizing the likelihood that a single locus-replacement event could achieve the evasion of vaccine-generated immunity.

Similarly, in the context of antibiotic usage, the influence these drugs have on the pathways of pneumococcal evolution will need to be considered in depth if we are to be  successful in the long-term control of pneumococcal infection.  Implementing treatment with individual new antibiotics at irregular intervals is unlikely to yield the results we seek.  A more comprehensive strategy that takes into consideration the ecology of pneumococcal acquisition of genes mediating antibiotic-resistance is probably going to be necessary.

References

Griffith F. The significance of pneumococcal types. J Hyg (Lond). 1928 Jan;27(2):113-59. PubMed PMID: 20474956; PubMed Central PMCID: PMC2167760.

The Oswald T. Avery Collection. http://profiles.nlm.nih.gov/ps/retrieve/Narrative/CC/p-nid/37.

Avery OT, Macleod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med. 1944 Feb 1;79(2):137-58. PubMed PMID: 19871359; PubMed Central PMCID: PMC2135445.

Croucher NJ, Harris SR, Fraser C, Quail MA, Burton J, van der Linden M, McGee L, von Gottberg A, Song JH, Ko KS, Pichon B, Baker S, Parry CM, Lambertsen LM, Shahinas D, Pillai DR, Mitchell TJ, Dougan G, Tomasz A, Klugman KP, Parkhill J, Hanage WP, Bentley SD. Rapid Pneumococcal Evolution in Response to Clinical Interventions. Science. 2011 Jan 28;331(6016):430-434. PubMed PMID: 21273480.

Coffey TJ, Enright MC, Daniels M, Morona JK, Morona R, Hryniewicz W, Paton JC, Spratt BG. Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Mol Microbiol. 1998 Jan;27(1):73-83. PubMed PMID: 9466257.

Feil EJ, Smith JM, Enright MC, Spratt BG. Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data. Genetics. 2000 Apr;154(4):1439-50. PubMed PMID: 10747043; PubMed Central PMCID: PMC1461021.

Lipsitch M. Vaccination against colonizing bacteria with multiple serotypes. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6571-6. PubMed PMID: 9177259; PubMed Central PMCID: PMC21091.

Lipsitch M. Bacterial vaccines and serotype replacement: lessons from Haemophilus influenzae and prospects for Streptococcus pneumoniae. Emerg Infect Dis. 1999 May-Jun;5(3):336-45. Erratum in: Emerg Infect Dis 1999 Sep-Oct;5(5):734. PubMed PMID: 10341170; PubMed Central PMCID: PMC2640786.