Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration

BACKGROUND: In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (τ) only up to a certain threshold cell density. Below this cell concentration (≤ 100 -1,000 CFU mL(-1 ); ≤ 27-270 CFU well(-1)) we observed an obvious increase in the τ scatter. RESULTS: Working with a food-borne E. coli isolate we found that τ values derived from two different microtiter platereader-based techniques (i.e., optical density with growth time {=OD[t]} fit to the sigmoidal Boltzmann equation or time to calculated 1/2-maximal OD {=t(m)} as a function of initial cell density {=t(m)[C(I)]}) were in excellent agreement with the same parameter acquired from total aerobic plate counting. Thus, using either Luria-Bertani (LB) or defined (MM) media at 37°C, τ ranged between 17-18 (LB) or 51-54 (MM) min. Making use of such OD[t] data we collected many observations of τ as a function of manifold initial or starting cell concentrations (C(I)). We noticed that τ appeared to be distributed in two populations (bimodal) at low C(I). When C(I )≤100 CFU mL(-1 )(stationary phase cells in LB), we found that about 48% of the observed τ values were normally distributed around a mean (μ(τ1)) of 18 ± 0.68 min (± σ(τ1)) and 52% with μ(τ2 )= 20 ± 2.5 min (n = 479). However, at higher starting cell densities (C(I)>100 CFU mL(-1)), the τ values were distributed unimodally (μ(τ )= 18 ± 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal τ distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL(-1)), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. CONCLUSION: These data argue that there is a dependence of growth rate on starting cell density.