Chromosome replication as a measure of bacterial growth rate during Escherichia coli infection in the mouse peritonitis model

Abstract

The efficacy of most antibiotics is dependent on active bacterial growth, yet little is known about the growth dynamics during infection. Therefore, means to measure in-host bacterial growth rate is of importance. Here, we use chromosome replication as readout for in situ bacterial growth rate during infection; obtained from a single biological specimen. We have applied two independent methods: quantitative PCR (qPCR) and fluorescence microscopy, to quantify the level of chromosome replication present during Escherichia coli propagation in the mouse peritonitis model. We find that the methods complement each other and allow for quantification of growth rate, both on a population average and on a single-cell level. We demonstrate the presence of heterogeneous growth rates within bacterial populations propagating during infection. Also, no growth cessation was observed during the apparent stationary phase in vivo, and, by comparison of growth dynamics at different anatomical sites, we demonstrate that E. coli is unlikely to grow independently intravascularly. These findings provide novel insight into bacterial growth during host infection, and underscore the importance of pinpointing the primary site of infection in septicaemia of unknown origin and ensuring antibiotic availability at this site.

Spheroplast-mediated carbapenem tolerance in Gram-negative pathogens

“…We conducted time-dependent killing experiments measuring both colony forming units (cfu/ml) and optical density (OD 600 ). Killing experiments for all isolates were conducted in supplemented Brain Heart Infusion (BHI+) broth under high inoculum/slow growth conditions (see Methods for details) to emulate the likely slow growth behavior during an infection (23). We chose a meropenem concentration (10 µg/mL), which is above the meropenem resistant breakpoint for Enterobacteriaceae [≥4 µg/mL], P. aeruginosa [≥8 µg/mL], and V. cholerae [≥4 µg/mL] (24, 25) and between 6.7 × and 625 × higher than the minimum inhibitory concentration (MIC) for each susceptible/non-resistant, non-carbapenemase producing isolate (Table 1).…”

Comparative activity of Ceftriaxone, Ciprofloxacin and Gentamicin as a function of bacterial growth rate probed by Escherichia coli chromosome replication in the mouse peritonitis model

“…We demonstrated correlation between ori:ter and bacterial cell size at all growth rates and the ability of ori:ter to predict the development in net bacterial population size. Moreover, in this recent observation of growth dynamics during host infection we found that growth rates were largely heterogeneous within the bacterial populations propagating both in the peritoneum and in the blood, respectively, throughout the length of infection (3). This finding is in consistency with previous reports of Staphylococcus aureus growth rate heterogeneity in cystic fibrosis sputum, as measured by isotope tracing (1).…”

“…1a), in vitro data from both versions of the strain were pooled for analysis. In regard to the Gentamicin treatment regimen, however, only wild-type ATCC 25922 was applied, as the Gentamicin MIC was increased by the presence of the non-removable Kanamycin cassette encoding Kanamycin phosphotransferase, used as clonal selection marker, in ALO 4783 (3, 29) (Table 1).…”

“…Studies of antibacterial activity are largely based on laboratory models, where balanced bacterial populations propagate rapidly in well-defined batch cultures with a finite quantity of life-sustaining nutrients. These in vitro models fail to mirror the true growth dynamics of bacterial pathogens taking place during infection in vivo , where growth in a bacterial population appears to be both slower and less homogeneous (1–3). The dependency upon active bacterial growth for most antibiotics to exert their effect is acknowledged and has been examined by other investigators, both in vitro and in vivo (4–11).…”

“…population mean ori:ter ~ 1) (20). By the use of two complementary methods for measuring ori:ter : quantitative PCR (qPCR) and fluorescence microscopy, we have been able to probe in situ growth rates of fluorescently labelled E. coli ATCC ® 25922™ both on a population average (by qPCR) and on a single-cell (by fluorescence microscopy) level during wide-spread infection in the mouse peritonitis model (3). We demonstrated correlation between ori:ter and bacterial cell size at all growth rates and the ability of ori:ter to predict the development in net bacterial population size.…”