Resistance to naphthoquinones

To examine the resistance of bacterial isolates to naphthoquinones, the growth of 13 bacterial strains from the predominant clade (clade 7) and seven low-abundant clades (clade 1, 6, 8, 9, 12, 17, 18) isolated from the ant hosts, and the 20 environmental bacterial isolates (10 Bacillus and 10 non-Bacillus) was compared using two naphthoquinones, respectively. As the fungal naphthoquinones are currently not purified and commercialized, the two naphthoquinones prepared for the experiment, plumbagin (de Paiva et al. 2003) and lapachol (Eyong et al. 2006), were those found in plants. The two naphthoquinones prepared for the experiment were dissolved in 30% dimethyl sulfoxide (DMSO) − 70% water solution. The naphthoquinone concentrations to be used for testing the bacterial resistance to this family of secondary metabolites were determined in a pilot test conducted as following; the naphthoquinones underwent serial dilutions (plumbagin: from 90–0.09 µg/mL; lapachol: from 257.5–0.5 µg/mL) from which the most suitable concentrations were picked based on the most obvious difference of growth rate among three randomly selected bacteria from the ant host and three from the environment. Based on these results, the concentrations of 45 µg/mL (plumbagin) and 64.5 µg/mL (lapachol) were used for further tests.

In this experiment, the bacterial isolates were first inoculated in LB medium at 20 °C (the mean annual temperature in Lianhuachi Research Center, where the infected ants were collected) overnight and then refreshed to the exponential phase with LB medium for 3 h. The bacterial concentration was adjusted to ~ 1.5 × 10⁸ cells/mL. Next, 10 μL of the bacterial suspension and 180 μL of Mueller − Hinton broth medium (Sigma-Aldrich) were added to either 10 μL of the naphthoquinone solution or 10 μL of the 30% DMSO − 70% water solution for the control. The growth of bacterial isolates at 20 °C was monitored by measuring the optical density at 600 nm (OD₆₀₀) with a Multiskan GO microplate spectrophotometer (Thermo Scientific) every hour for 12 h. Each combination of bacterial isolate and naphthoquinone or control was replicated twice.

The resistance index of each bacterial isolate was calculated by the following formula: [OD_{naphthoquinone} – OD_DMSO]/[OD_{naphthoquinone} + OD_DMSO]. The resistance index was analyzed using a linear mixed model with the resource (Bacillus from the ant host, Bacillus and non-Bacillus from the environment) as the fixed effect, the bacterial isolate and experimental replication as random effects, and growth time (5 − 12 h) as a nest effect. The significance of the fixed effect was examined by the likelihood ratio test with the model removing the fixed-effect term. Post hoc tests were undertaken using Tukey's all-pair comparisons. The model building and hypothesis tests were conducted using the "lme4" and "multcomp" packages in R.