Importance of Bacterial Inoculum Size

Integral to the design of the original microbial endocrinology experiments was a commitment to use a bacterial inoculum that accurately reflected the infectious dose encountered in vivo. Therefore, the starting inocula used were low, typically these were around 10'-102 CFU/ml, which was intended to reflect the numbers of pathogenic bacteria likely to be present at the start of an infection (Lyte and Ernst 1992, 1993; Freestone et al. 1999, 2002, 2003, 2007a, b, c). This use of low bacterial numbers is in direct contrast to the majority of in vitro bacterial virulence studies which generally utilise many log orders higher inocula in their experiments. Indeed, several microbial endocrinology-related studies, mainly for technical reasons, have examined responses to catecholamines using high cell density cultures (around 108 CFU/ml); the authors have then used the data obtained in these studies to make inferences to in vivo scenarios, where the infecting bacteria are likely to be in much lower numbers (Sperandio et al. 2003; Vlisidou et al. 2004; Walters and Sperandio 2006), but are such extrapolations really valid? It has been observed that at low cell densities (102 CFU/ml), Yersinia enterocolitica shows no growth response to epinephrine, and furthermore epinephrine can actually antagonize Yersinia responses to other catecholamines (Fig. 16.1, Freestone et al. 2007a); however, the same Y. enterocolitica when analysed at high cell density (108 CFU/ml) are able to use a similar concentration of epinephrine to specifically acquire iron from transferrin (Freestone et al. 2007a). As it has already been determined that bacterial growth induction in low density cultures is mainly due to catecholamine-assisted iron uptake (Freestone et al. 2000), this failure of the Y. enterocolitica low density cultures to respond to epinephrine is very difficult to explain unless we can hypothesise that bacteria may in fact use the same catecholamines in different ways in a manner which is dependent upon their population density.

Additional experiments have demonstrated that the population density of a bacterial culture can greatly influence the apparent specificity of catecholamine responsiveness. Freestone et al. (2007a) used serum-SAPI medium to examine the growth responses of E. coli O157:H7, S. enterica and Y. enterocolitica to various catecholamines over an 8-log dilution curve (Fig. 16.2). It can be seen that the effect of catecholamines on bacterial growth in a serum-based medium becomes evident at low (<104 CFU/ml) cell densities with the greatest differences observed at very low (<102 CFU/ml) cell densities. Although we see in Fig. 16.2 that E. coli O157:H7 and S. enterica are both able to respond to epinephrine at very low population densities, which reflect the bacterial numbers that are likely to be present at the initial stages of an infection, it is apparent that there is an order of catecholamine preference evident, with growth responses to norepinephrine and dopamine being at least a log-fold greater than those of epinephrine. It seems obvious based upon these observations and the Y. enterocolitica epinephrine response data (Freestone et al. 2007a) that caution should be exercised before assuming that conclusions reached from in vitro observations of microbe-host catecholamine responses in high density cultures can be directly extrapolated to the low population numbers typically present during the initial stage of an infection (Tarr and Neill 2001).

It must also be appreciated that when using host-like media such as serum-SAPI, the use of large numbers of bacteria in the inoculum can lead to problems with evaluating a response to a specific neuroendocrine hormone due to the cell mass overwhelming the media's bacteriostatic potential. For example, Belay et al. 2003 reported differential effects of catecholamines on the growth of several gram negative bacterial species which appeared to be in contrast to those of previous publications (Lyte and Ernst 1992, 1993). In these experiments, the initial bacterial inoculum was so large that no lag phase occurred in the serum-based medium and maximal growth was achieved by both control and catecholamine-supplemented

NE+E100 NE+E200 NE+E300 Control

Fig. 16.1 The ability of epinephrine to inhibit Y. enterocolitica growth induction by norepinephrine and dopamine. Y. enterocolitica (a and b) and E. coli 0157:H7 (c and d) were inoculated at approximately 102CFU/ml into duplicate 1 ml aliquots of serum-SAPI containing the combination of catecholamines shown, and incubated for either 40 h (Y. enterocolitica) or 18 h (E. coli 0157:H7), and enumerated for growth (CFU/ml) as per standard technique (Freestone et al. 2000). The results shown are representative data from four separate experiments; data points typically showed variation of no more than 3%. Black bar catecholamine/catecholamine combination only, light grey bar catecholamine/catecholamine combinations plus 100 |.iM Fe(N03)r Key: NE norepinephrine. Dop dopamine, and Epi epinephrine, (black bar). NE 50[M NE. NE+E100 50^M NE plus 100[M Epi. NE + E200 50[M NE plus 200[M Epi. NE+E300 50[M NE plus 300[M Epi. Dop 50[M Dop. Dop + ElOO 50nM Dop plus 100|.iM Epi. D + E200 50|.iM Dop plus 200|.iM Epi. Dop + E300 50|.iM Dop plus 300|.iM Epi. The results shown are representative data from three separate experiments; data points typically showed variation of no more than 3%. This figure was taken with permission from Freestone et al. (2007a)

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1

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Dop Dop+E100 Dop+E200 Dop+E300

Dop Dop+E100 Dop+E200 Dop+E300

Dop Dop+E100 Dop+E200 Dop+E300 Control

NE+E100 NE+E200 NE+E300 Control

Dop Dop+E100 Dop+E200 Dop+E300 Control

Fig. 16.1 The ability of epinephrine to inhibit Y. enterocolitica growth induction by norepinephrine and dopamine. Y. enterocolitica (a and b) and E. coli 0157:H7 (c and d) were inoculated at approximately 102CFU/ml into duplicate 1 ml aliquots of serum-SAPI containing the combination of catecholamines shown, and incubated for either 40 h (Y. enterocolitica) or 18 h (E. coli 0157:H7), and enumerated for growth (CFU/ml) as per standard technique (Freestone et al. 2000). The results shown are representative data from four separate experiments; data points typically showed variation of no more than 3%. Black bar catecholamine/catecholamine combination only, light grey bar catecholamine/catecholamine combinations plus 100 |.iM Fe(N03)r Key: NE norepinephrine. Dop dopamine, and Epi epinephrine, (black bar). NE 50[M NE. NE+E100 50^M NE plus 100[M Epi. NE + E200 50[M NE plus 200[M Epi. NE+E300 50[M NE plus 300[M Epi. Dop 50[M Dop. Dop + ElOO 50nM Dop plus 100|.iM Epi. D + E200 50|.iM Dop plus 200|.iM Epi. Dop + E300 50|.iM Dop plus 300|.iM Epi. The results shown are representative data from three separate experiments; data points typically showed variation of no more than 3%. This figure was taken with permission from Freestone et al. (2007a)

E. coliO157:H7

3 105

104 103 102 101

E. coliO157:H7

Dilution Factor S. enterica

Dilution Factor S. enterica

Dilution Factor

Y. enterocolitica

Dilution Factor

Y. enterocolitica

Dilution Factor

Fig. 16.2 Bacterial population density influences catecholamine specificity. Histograms (a)-(c) shows the growth response of varying inoculum size on the specificity of growth response to catecholamines norepinephrine (NE), epinephrine (Epi) and dopamine (Dop). Cultures were diluted in tenfold steps into serum-SAPI medium, incubated for either 18 h (E. coli O157:H7 and S. enterica) or 40 h (Y. enterocolitica), and enumerated for growth (CFU/ml) as described in Freestone et al. (2007a). The inoculum size of the E. coli culture was 5 x 108 CFU/ml. The results shown are representative data from four separate experiments; individual data points showed variation of no more than 5%. White bar no additions (control), light grey bar 50 |iM NE, black bar 100 |iM Epi, diagonal hatch 50 |iM Dop. This figure was taken with permission from Freestone et al. (2007a)

Dilution Factor

Fig. 16.2 Bacterial population density influences catecholamine specificity. Histograms (a)-(c) shows the growth response of varying inoculum size on the specificity of growth response to catecholamines norepinephrine (NE), epinephrine (Epi) and dopamine (Dop). Cultures were diluted in tenfold steps into serum-SAPI medium, incubated for either 18 h (E. coli O157:H7 and S. enterica) or 40 h (Y. enterocolitica), and enumerated for growth (CFU/ml) as described in Freestone et al. (2007a). The inoculum size of the E. coli culture was 5 x 108 CFU/ml. The results shown are representative data from four separate experiments; individual data points showed variation of no more than 5%. White bar no additions (control), light grey bar 50 |iM NE, black bar 100 |iM Epi, diagonal hatch 50 |iM Dop. This figure was taken with permission from Freestone et al. (2007a)

cultures in less than 12 h (Belay et al. 2003). However, when the authors later conducted similar experiments using much lower inocula (O'Donnell et al. 2006), they observed a lag phase that is more characteristic of this bacteriostatic medium

(Lyte and Ernst 1992, 1993; Freestone et al. 1999, 2002, 2003, 2007a, b, c); they also reported an increase in bacterial response to norepinephrine which had not been observed in the previous work. It is also paramount that, given the inherent variability in bacterial culture viability, the optical density measurements which are typically used to estimate CFU/ml for culture initiation should always be confirmed by performing plate counts to determine the numbers of bacteria that were actually in the inocula used.

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