2.1. Effect of Antibiotic on Tobacco Shoot Growth and Oxidative Stress Injury
Supplementation of growth medium with 250 mg L−1 timentin had a growth-suppressing effect on tobacco in vitro shoot culture. After three weeks of cultivation, the average fresh weight (FW) of the control tobacco shoots (TC) was estimated to be 164.7 ± 3.3 mg (Figure 1, Appendix A Table A1). The FW of the shoots cultivated on medium containing timentin (TA) was reduced by 29 ± 4% (p = 1.5 10−7) compared to the TC shoots.
An analysis of the membrane lipid oxidative injury of the TC shoots revealed significant variation in malondialdehyde (MDA) concentration during the three weeks of the propagation cycle (Figure 2; Appendix A Table A1). The highest value was detected at day four after transfer to fresh medium and was estimated at 95.6 ± 2.6 nmol g−1 FW. The increased MDA accumulation in the control shoots was likely a consequence of the combined effect of stress associated with tissue senescence and shoot injury during the transfer to the fresh medium, which was followed by adaptation and active growth on the fresh medium. This resulted in an ~1.3-fold (p = 4 × 10−6) decrease in MDA concentration at the end of the first week of cultivation (day 7) and a subsequent gradual increase in MDA concentration during the course of culture senescence over the remaining two weeks of the propagation cycle. However, the TA shoots maintained on the antibiotic supplemented medium did not follow this cycle and showed 1.2–1.6-fold (p < 0.014) higher levels of MDA than the TC shoots over the entire period of the propagation cycle (Figure 2), which could be a direct consequence of antibiotic-induced cytotoxic effects.
Interestingly, after transfer to the medium without antibiotic (PA), a consistent residual negative effect of antibiotic treatment on shoot growth vigor and stress level was observed for at least several passages used in the experiments. Cumulative data from several of these experiments revealed that the growth vigor of the shoots was only partially restored, and that the accumulation of biomass remained significantly lower (9 ± 3%, p = 0.013) compared to TC (Appendix A Table A1). Similarly, the level of MDA accumulation was reduced compared with TA and showed a significant decrease during the first week after transfer to fresh medium. Nevertheless, it remained 1.1–1.3-fold (p < 0.033) higher compared to the control group over the remaining two weeks of the propagation cycle.
2.2. Analysis of Bacterial Diversity in Tobacco In Vitro Shoot Samples
To investigate bacterial diversity in tobacco shoot culture and to assess the effect of antibiotic treatment on bacterial diversity, DNA extracted from shoots of the TC and PA experimental groups was subjected to 16S rRNA gene amplicon analysis using the Ion Torrent high-throughput sequencing platform.
The overall number of high-quality mapped sequences with 224–225 bp read length was similar for the TC and PA libraries (1,027,576 and 1,175,938, respectively) but varied up to ~3.5-fold for different DNA preparation methods and primer pairs specific to distinct 16S rRNA gene regions (Figure 3, Appendix A Table A2). The proportion of bacterial sequences varied from 10% to 29% for the different DNA extraction methods (Figure 3a). Independent of the experimental group, consistently higher content of bacterial operational taxonomic units (OTUs) (>25% of all reads) was detected for samples prepared with DNA extraction methods described by Doyle [40] and Ding et al. [41]. For the TC shoots, similar results were also obtained using the DNA extraction method described by Li et al. [42]. The remaining two methods resulted in a consistently lower proportion of the bacterial OTUs. Bacterial DNA enrichment by selective organelle lysis using SDS and NaCl as previously described by Wang et al. [43] for preparation of microbial metagenomic libraries of tropic tree Mallotus nudiflorus did not increase bacterial OTU content for the tobacco shoot samples as compared to other DNA extraction methods, which suggests that this approach requires further optimization.
The proportion of sequences assigned to the bacterial and tobacco plastid or mitochondrial 16S rRNA also varied depending on the PCR amplification region and/or specificity. Two primer pairs specific to the V4 and V8 regions generated the largest number of reads mapped as bacterial OTUs and the largest proportion of the bacterial OTUs compared to the overall number of reads (Figure 3b). Meanwhile, the overall amplification efficiency of the V6–7 and V2 primers was 2- to 4-fold lower, and the resulting proportion of bacterial sequences was comparable to that of V8. The V3 primers generated a comparable number of reads in total to the V8 region, but these reads contained the lowest proportion of bacterial sequences. Notably, for all primer sets, a consistently lower ratio of bacterial to plastid/mitochondrial OTUs was obtained for the PA sample than for the TC sample which could be a result of lower yields of bacterial DNA due to lower bacterial density in the antibiotic-treated shoot sample.
The 16S rRNA region-specific primer propensity for specific taxonomic groups was assessed using family-level data to avoid bias due to the limited accuracy of genus or species identification using short-read sequences which could lead to underestimation of primer specificity. The association among the OTUs obtained using different primer pairs was mapped on the UpSet plot using cumulative data from both experimental groups (Figure 4). Among the 59 family-level OTUs, 22% were detected by all primer pairs and were represented by 96% or 70% of all bacterial reads when the dominant Mycobacteriaceae family was included or excluded from the analysis, respectively. No singleton families were detected using V2-specific primers and 26 (42% of all families) singleton families were detected by the remaining four primer pairs.
Previously, variation in the informative power of the same 16S rRNA region-specific primers has been demonstrated in applications to human intestinal and environmental microbiota samples [44–46]. In our study, the read abundance for the same OTU also varied considerably among the primer pairs (Figure 5). For example, the most abundant Mycobacteriaceae family was represented by 37%, 18%, and 39% of the total number of reads using V4, V6–7 and V8 primers, while only 6% and <1% of the total number of reads were assigned to the family for the V2 and V3 primer data sets, respectively. In another instance, the distribution of reads assigned to the Paenibacillaceae family among the V3, V4, and V8 data sets was 15%, 38%, and 44%, respectively, while V2 and V6–7 represented only 3% and <1% of the total number of reads, respectively. Regardless of the apparent taxa-specific amplification efficiency among the primer pairs, the hierarchical cluster analysis of the data did not reveal a consistent distribution of taxonomic groups among the primer data sets (Figure 5), possibly due to variation of primer specificity at a lower taxonomic level or the bias introduced by variation of the taxa abundance.
2.3. Antibiotic Effect on Bacterial Diversity in Tobacco Shoot Culture
Although both the TA and PA samples had a similar overall number of reads and reads mapped to bacterial OTUs, the number of unique OTUs was reduced from 153 to 27 upon antibiotic treatment (Appendix A Table A2). Beta-diversity analysis of the microbiome data sets using NMDS with Bray–Curtis dissimilarity matrix represented variation between the two tobacco shoot experimental groups on the first coordinate (Figure 6).
Among the six phyla detected in the tobacco shoot microbiome, Actinobacteria was dominant in both experimental groups and was mainly represented by the order Actinomycetales, including 17 and 3 families for the TC and PA experimental groups, respectively (indicated in a blue font in Figure 5; Supplementary Material Table S1). Actinobacteria included the most prevalent family in the data set, Mycobacteriaceae, which represented 81% and 98% of mapped bacterial reads for TC and PA, respectively. Microbacteriaceae represented another antibiotic treatment-enduring Actinobacteria that became the second most abundant OTU with a relative abundance of 1.6% in the antibiotic-treated shoot sample, while Propionibacteriaceae and Acidimicrobiaceae were detected only at marginal levels. For OTUs in the latter family, sequence comparison showed 98% sequence similarity to Mycobacteriaceae, which could also imply inaccuracy of the sequence assignment. A similar assumption could be drawn for the assignment of the Thermolithobacteraceae family of the phylum Firmicutes. Meanwhile, Bacillaceae and Paenibacillaceae were the most abundant among the remaining four families of Firmicutes representing 3% of mapped bacterial reads in the control shoots. In addition, Staphylococcaceae and Streptococcaceae were detected at low abundance but by three primer pairs each. The abundance of all Firmicutes was largely reduced (<0.1% of mapped reads) upon antibiotic treatment.
In the control shoots, 1.6% of mapped reads were assigned to five families of Bacteriodetes, mainly represented by Sphingobacteriaceae, Chitinophagaceae, and Flavobacteriaceae, but all of them were undetectable in the antibiotic-treated sample. Among the Proteobacteria including 8% and 0.5% of mapped bacterial reads of the TC and PA, respectively, class Alpha-proteobacteria included 10 families of order Rhizobiales representing many well-known beneficial plant-associated bacteria [47] and Caulobacteraceae and Sphingomonadaceae families including common environmental bacteria [48,49]. In the antibiotic-treated shoots only Caulobacteraceae and Bradyrhizobiaceae were detected at 4- and 10-fold reduced abundance levels compared to control shoots, respectively. Interestingly, the families Kopriimonadaceae, Rhodobacteraceae, and Rhodospirillaceae, which includes species of common environmental and aquatic bacteria, were detected at low abundance only in the antibiotic-treated shoots.
The Desulfovibrionaceae family of class Delta-proteobacteria was detected at low but similar abundance in both TC and PA shoots. However, more proliferous Beta-proteobacteria and Gamma-proteobacteria (including 5 and 7 families, respectively) were also largely reduced upon antibiotic treatment. Among the Beta-proteobacteria were notable Alcaligenaceae and Methylophilaceae which include nitrifying and methylotrophic environmental bacteria [50,51]. Meanwhile, the Enterobacteriaceae and Pseudomonadaceae families of Gamma-proteobacteria represent numerous plant endophytes or pathogens [52]. In addition, two families of the PVC superphylum, Planctomycetes and Verrucomicrobia, which include common soil and plant root-associated bacteria [53] were both detected at low abundance in the control shoots.
2.4. Isolation of Actinobacteria and Antibiotic Resistance Assessment
To further characterize the Actinobacteria dominant in the in vitro tobacco shoot culture, an extract of antibiotic-treated shoots was plated on Loewenstein–Jensen growth medium. The isolate obtained from bright yellow colonies formed after 6–8 weeks of incubation showed 98% identity to the M. cookii strain ATCC 49,103 (GenBank accession NR_114661.1) [54] based on the 1407 nt fragment of 16S rRNA. The isolate also sustained similar growth properties on Actinobacteria Isolation Agar, which was used for later cultivation and antibiotic resistance tests. The isolate showed resistance to timentin at the concentration used for the tobacco shoot treatment (250 mg L−1) and chloramphenicol at 30 mg L−1 (Figure 7); however, the growth of the isolate was suppressed by rifampicin at 25 mg L−1, which suggests this antibiotic could be used to eliminate mycobacteria from tobacco in vitro cultures.