Date Published: March 20, 2014
Publisher: Public Library of Science
Author(s): Alice Gerlini, Leonarda Colomba, Leonardo Furi, Tiziana Braccini, Ana Sousa Manso, Andrea Pammolli, Bo Wang, Antonio Vivi, Maria Tassini, Nico van Rooijen, Gianni Pozzi, Susanna Ricci, Peter W. Andrew, Uwe Koedel, E. Richard Moxon, Marco R. Oggioni, Bruce R. Levin.
The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.
Streptococcus pneumoniae, one of the major human bacterial pathogens, is also part of the normal upper respiratory tract flora, where nasopharyngeal colonisation with one or more strains often lasts weeks to months with seasonal peaks in late winter , . Carriage of S. pneumoniae (pneumococci) may result in disease as the consequence of contiguous spread from the nasopharynx to other sites in the upper or lower respiratory tract causing, for example, otitis media or pneumonia. More rarely, there is hematogenous dissemination of pneumococci resulting in septicaemia and metastatic disease such as meningitis , –. In experimental models of pneumococcal infection, the challenge dose required to induce disease is dependent on the route of infection, the genetic background of the host and the virulence of the infecting strain  and may vary from a very few to millions of organisms . Following intravenous inoculation of mice with laboratory grown pneumococci, a hallmark of experimental bacteraemic infections is the rapid and efficient clearance of most of the inoculated bacteria –. In non-immune rodents, major factors mediating this clearance are splenic macrophages and complement mediated opsonisation –. A challenge dose of about one million virulent, encapsulated pneumococci is generally needed to induce bacteraemia in about half of challenged animals (the effective dose or ED50) and which is the dose generally used to address investigations into the early events shaping an infectious process.
We have investigated the pathogenesis of pneumococcal bacteraemia following intravenous inoculation of mice with three isogenic clones (variants). In our model, the infection followed the classic, three phase pattern in which a majority of pneumococci are cleared in the first minutes post-challenge. This leads to an “eclipse phase” of several hours in which bacterial numbers decline further or are undetectable. This is followed by the emergence of sustained and high density bacteraemia in a proportion of the challenged animals , . By analysing the survival in the blood of three isogenic variants of S. pneumoniae, we observed that the majority of blood cultures arose from only one of the three variants. We used a statistical model to characterise the infection dynamics in which the number of bacteria starting the infection in each invasion event is w and the number of times this happens is k . From the model, we could infer that the number of bacteria at the origin of infection is below 2 (w = 1). Thus, it follows that bacteraemia was generated by either (a) a single bacterium establishing a population in the blood in a single invasion event or (b) several bacteria each of which independently established a population in distinct invasion events. The probability of (b) is small (about 5% in our data, because the probability of two or more invasion events occurring is about 5%). Genome sequencing provided genetic evidence in 4/6 cases that monoclonal bacteraemia did actually start from a single bacterial cell (w = 1) confirming the first statement. For the remaining 2/6 cases we could not determine w = 1 by genome sequencing as we could not distinguish several invasions of a single bacterial variant from one invasion of several cells of the same variant without any SNPs. More complex is the experimental observation of invasive events. For this we could document bacteraemia in mice with previous negative blood samples (k≥1) and in other mice the increase of variants in serial blood samples (k>1). Since after the first 24 h the observed numbers of both these types of invasion events are similar, this strongly favours the occurrence of polyclonal infections resulting from independent, not cooperative action. In the case of H. influenzae it had been hypothesised that the single cells giving rise to the monoclonal infection might be selected by within-host evolution . Our work now tests this hypothesis by whole genome sequencing. The data show in two cases absence of any SNPs and in four cases SNPs that apparently do not indicate selection for virulence. Despite the low numbers, it suggests that the single cells at the origin of infection apparently have no advantage (higher virulence) over the other cells in the population. Such results show that the bacteria in the challenge dose act independently to give rise to infection, that each has a similar probability of causing infection and that a dose near the LD50, a single cell may initiate disease. These criteria satisfy the theory of independent action , , , . As such our investigation provides strong evidence that the single founding cell of an invasive infection is the result of a stochastic event. However, it must be emphasised that epigenetic variations would have eluded our genetic and genomic analysis.