Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology

Abstract

Aim: To develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP).

Methods and results: Seven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM).

Conclusions: MAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 μg ml^-1 Mycobactin J.

Significance and impact of the study: This is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.

Keywords: Mycobacterium avium subsp. paratuberculosis; egg yolk; growth supplements; medium composition optimization; response surface methodology; solid culture.

FIGURE 1

Growth curves for Mycobacterium avium subsp. paratuberculosis (MAP) 796 PSS on 35 different formulations of agar (runs) tested during the fractional factorial design experiment. (a) Runs 1–12, (b) 13–24 and (c) 25–35. Figure legends list agars from the best to the worst growth in each case. Details of the composition of each agar (run) are provided in Table S1

FIGURE 2

Pareto charts used for manual selection of significant factors after fractional factorial design experiment: (a) response 1 (number of days until first Mycobacterium avium subsp. paratuberculosis (MAP) colonies became visible) and (b) response 2 (average cfu/100 μl inoculum after 37 days of incubation). On both graphs, A = Tween 80, B = Taurocholic acid, and F = egg yolk. Orange bars indicate a positive effect of the factor, or combination of factors, on MAP growth (i.e. MAP colonies became visible earlier), while blue bars indicate a negative effect (i.e. MAP colonies took longer to become visible)

FIGURE 3

Growth curves for Mycobacterium avium subsp. paratuberculosis (MAP) 796 PSS on 40 different formulations of agar (runs) tested during the Response Surface Methodology experiment. Agars were supplemented with either 10% OADC (a–c) or 10% ADC (d–f) and no egg yolk (a, d), 5% egg yolk (b, e) or 10% egg yolk (c, f). Figure legends list agars from the best to the worst growth in each case. Details of the composition of each agar (run) are provided in Table S2

FIGURE 4

Mycobacterium avium subsp. paratuberculosis (MAP) cultures (10 μl) streaked on (1) Herrold's egg yolk mycobactin J agar (HEYM), (2) the optimized solid medium and (3) Middlebrook 7H9 agar with OADC and Mycobactin J, demonstrating more luxuriant growth on 2 after 27 days of incubation compared to 1. (a) MAP ATCC 19698, (b) MAP B4, (c) MAP 796 PSS and (d) MAP VIAS‐Crohn's disease