Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates

Background Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. Methods In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. Results The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). Discussion Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

We generated two groups, one with data from all clinical isolates and the other with data from all 140 environmental isolates. Statistical differences between clinical and environmental isolates in the 141 metabolization of butyric acid and propionic acid were evaluated by means of 95% family-wise 142 comparison of group means (Tukey contrast test) of the parameter A on specific wells using the 143 function "opm_mcp" within the opm R-package. A p value less than 0.05 was considered to be 144 statistically significant.
145 Whole genome sequencing of MAH isolates 146 Genomic DNAs were extracted from the MAH isolates as described previously (Lewin et al. 147 2003). Whole genome sequencing (WGS) was performed using Illumina MiSeq 300 bp paired-148 end sequencing, yielding a coverage that exceeded 100x. The NGS QC tool kit was used to 149 assess the quality of the data reads, which was set as reads with a minimum of 70 % of bases 150 having a phred score greater than 20 (Patel & Jain 2012). De novo assembly of the resulting 151 reads into multiple contigs was performed using CLC Genomics Workbench 8.0 (CLC bio, 152 Aarhus, Denmark) and contigs annotation was done using RAST (Aziz et al. 2008).
153 Determination of the maximum common genome and of the accessory genome 154 We determined the maximum common genome (MCG), comprising those genes present in all of 155 the ten MAH genomes, as reported previously (von Mentzer et al. 2014). All these genes were 156 then extracted from all genomes, concatenated and aligned. The resulting alignment was used to 157 generate a clustering tree using RAxML 8.1 (Stamatakis 2014). 158 For determination of the accessory genome we applied the PanGenome Pipeline -Roary. After 159 determination of the accessory genome of the ten MAH genomes and its distribution within . Although our study did not reveal any clear distinction between clinical or 257 environmental MAH isolates at the level of the whole genome, we observed differences between 258 clinical and environmental isolates with regard to substrate utilization. The most intriguing 259 difference is that the two fatty acids butyric acid and propionic acid are metabolized more by the 260 environmental than by clinical isolates. 261 We observed no difference in the presence / absence of genes associated with butyric or 262 propionic acid pathways among the group of clinical and the group of environmental MAH 263 isolates. The SNPs analysis of the genes involved in the pathways revealed that no SNPs were 264 associated with clinical or environmental origin of the MAH isolates. These evidences suggest 265 that the metabolic differences observed among clinical and environmental MAH isolates might 266 be due to difference in gene regulation. However, we screened all the genes that up to now have 267 been associated with the pathways of interest. We can speculate that there might be additional 268 genes, of unknown function, that might play a role in the above pathways. 269 The analysis of the accessory genome revealed that none of the genes specific for the clinical or      Additives used for each PM plates As additive are usually provided nutrient that are absent to the PM minimal media, but present in a standard MAH growth conditions. We used additives to make a complete minimal medium but omitted anything that could act as a source of the substrates of interest (for example, we did not include nitrate additives in the nitrogen source plates).
1 Table 2 Additives used for each PM plates. As additive are usually provided nutrient that are absent to the PM minimal media, but present in a 2 standard MAH growth conditions. We used additives to make a complete minimal medium but omitted anything that could act as a source of the 3 substrates of interest (for example, we did not include nitrate additives in the nitrogen source plates  Figure 1 Heatmap showing the 20 substrates that were differently metabolized by the ten MAH isolates analyzed in this study.
The color key scale for each substrate is based on dye reduction quantified by Omnilog units.  Clustering of the 10 MAH isolates.
The tree was generated using RAxML 8.1. The alignment comprised 1,658 genes constituting the maximum common genome of our ten MAH isolates. Two reference strains were also included (MAH 104 and MAH TH135). The genome sequence of M. avium subsp.
paratuberculosis K10 (Accession Number: AE016958) was used as outgroup. Isolate origin is also represented by blue for clinical origin and orange for environmental origin. The percentage of trees in which the associated taxa clustered together is shown adjacent to the branches.