Maria Schei Haugan:
Examination of bacterial growth rate during host infection and its importance for antimicrobial treatment

Date: 31-01-2019    Supervisor: Anders Løbner-Olesen

Escherichia coli is the leading cause of bacteraemia caused by Gram negative bacteria and the overall leading cause of urinary tract infection in humans. Bacterial infections arise when bacteria are able to evade the immune system of the host and access areas where they can proliferate. Being metabolically active is a necessity for the production of virulence factors, and most antibiotics in clinical use depend on active bacterial growth to exert their effect. However, little is known about the growth dynamics taking place during infection in the host (in vivo). Despite several techniques to measure in vivo bacterial growth rate having been proposed over the past decades, no gold standard method exists. The aim of this PhD thesis was to explore the use of molecular biology methods to measure the mode of the E. coli chromosome replication apparatus as readout of bacterial growth rate during host infection. The thesis is based on three papers.

In paper I, we demonstrate the applicability of bacterial chromosome replication status as a means to measure in situ growth rates of E. coli propagating in the peritoneum and in the blood during experimental disseminated infection in mice. From a combination of two complementary methods (real-time quantitative PCR [qPCR] and fluorescence microscopy) for differential chromosomal origin (oriC) and terminus (terC) copy number quantification (ori:ter) in fluorescently labelled E. coli, we were able to measure in vivo bacterial growth rate both on a population average and on a single-cell level. We demonstrate in this model: (i) that the bacterial populations propagating during infection are continually heterogenic (i.e., constituted by cells growing at different growth rates), (ii) that a complete cessation of growth does not occur, and (iii) that E. coli does not grow independently in the bloodstream during septicaemia.

In paper II, we extended the approach of probing bacterial growth rate from differential genome origin and terminus copy number quantification into exploring its potential in predicting antibiotic effect as a function of pretreatment in situ bacterial growth rate in the same experimental infection model. Here, we demonstrate that the activities of the β-lactam antibiotic ceftriaxone and the aminoglycoside antibiotic gentamicin were highly dependent on active bacterial growth. The fluoroquinolone ciprofloxacin, however, was less sensitive to bacterial growth rate, as the activity remained largely unchanged in rapid, compared to slow, bacterial growth rate treatment induction in vivo. Moreover, by analysis of posttreatment ori:ter we demonstrate that ceftriaxone and gentamicin induced preferential elimination of rapidly growing cells, whereas this was not evident for ciprofloxacin.

In paper III, we included hospitalised patients with E. coli bacteriuria in a clinical study to evaluate the use of differential genome quantification as a measure of growth rate of bacteria propagating in the human urinary tract. We were able to track bacterial growth rates in patients both with and without (i.e., asymptomatic bacteriuria) symptoms of urinary tract infection (UTI) for up to four consecutive days. We observed active bacterial growth in the majority of the urine samples. There were somewhat higher bacterial growth rates in patients with UTI (compared to patients without) and in patients with invasive infection (compared to patients without), suggesting that active bacterial growth could be a factor contributing to pathogenicity. However, these observations warrant future evaluation in a larger cohort.

In summary, the findings reported in these papers provide insight into the bacterial growth dynamics taking place during host infection and demonstrate the contribution of bacterial growth rate to antibiotic effect in vivo.