QUANTIFICATION AND MODELING OF AGGREGATE BACTERIAL GROWTH IN SUSPENSION: IMPLICATIONS FOR BACTEREMIA

Abstract

Bacteremia is a central event in the pathogenesis of sepsis. While much is known clinically of blood stream infections, little is known of bacterial life in the bloodstream. We have observed that Klebsiella pneumoniae in liquid media tend to grow in multicellular aggregates, or flocs. Hypothesizing that bacterial floc formation could have significant importance to the proliferation and clearance of bacteria from the bloodstream, we studied floc formation using an in vitro model. K. pneumoniae strain 43816 (a mouse pathogen) was grown in minimal media with 1% glucose at 37°C on a platform orbiting at 100 rpm. Floc volume was quantified with a Coulter counter using 5- to 400-femtoliter particle windows (with the average volume of a single bacterium ~ 5 fL). Growth dynamics were modeled using both log-linear statistical and partial differential equation (PDE) mechanistic models. Confocal laser scanning microscopy (CLSM) was used to study floc structure. Under the conditions studied, flocs from Kp 43816 formed quickly. Log-linear modeling revealed the predicted number of flocs of volume v (in fL) at time t (in minutes) was 10^(5.49+0.19t-(2.10 log(v))), with the p value for each parameter < 0.0001. The PDE model captured significantly more detail of growth and suggested that aggregate formation was due to bacterial proliferation rather than bacterial collisions. Digital image analysis of CLSM cross sections indicated that ~25% of floc volume was comprised of bacteria with the remaining volume being extracellular matrix. Analysis of 3 clinical blood isolates revealed similar behavior. CONCLUSION: K. pneumoniae, a common bloodborne pathogen, forms multicellular structures when grown in conditions mimicking growth in blood. These structures may achieve a size too large either to be phagocytosed or to pass through some capillaries. Future studies of bacterial transit in flowing blood should consider multicellular, and not just singlecellular, architecture.