General genomic characteristics of S. maltophilia strains
Genomic characterization of 22 clinical and 19 environmental S. maltophilia isolates was performed as shown in Fig. 1A and 1B. The genome size of S. maltophilia ranged from 4.4 to 5.0 Mb in the environmental-derived isolates and 4.4 to 5.1 Mb in the clinical ones. Additionally, the G + C content of environmental S. maltophilia spanned a wide range of 65.00 ∼ 67.5%, and the G + C content of clinical strains was concentrated at 66.00-67.00%. The CDS content in the environmental isolates was concentrated between 3900 and 4300 and was relatively stable, whereas it was much more dispersed in the clinical-derived strains with CDS content ranging from 3800 to 4700, probably because those strains showed genetic variability and diversity in different beating hosts. No remarkable differences were observed between environmental and clinical isolates in terms of genome size, CDS number, and G+C content (Fig. 1C). Although previous studies have shown that bacterial genome size is related to the selection pressure of the survival environment (Yu et al. 2018), no significant distribution was observed in the 42 isolates, which might be related to the fact that those isolates selected in this study were from multiple biomes. A literature reported significant differences in genome size and CDS number depending on the origin of Streptomyces strains (Tian et al. 2016).
Core gene and pangenetic analysis
In general, pan-genomes can be divided into open pan-genomes and closed pan-genomes according to their characteristics (Wang et al. 2020). The open or closed features of the pan-genome reflect genomic diversity and ability of the strain to adapt to environmental changes and acquire new traits through the transfer of genetic material under environmental selection (Li et al. 2021). As shown in Fig. 2A, the size of the pan-genome increased with the addition of genomes, while the size of the core genome decreased as the genome number augmented. The representing functional relationship of the fitted curve was " n = 3655.62.479*N -0.302", wherein α value 0.302 (α<1) implied that S. maltophilia had an open pan-genome. The size of the core genome was progressively closer to the steady-state than the pan-genome, implying that genus-level isolates had a sizable potential to ingest foreign genetic material and boost genetic diversity through other evolutionary mechanisms including mutation and horizontal transfer. As a result, S. maltophilia might change its environment by gaining and losing accessories and unique genes.
The pangenome contains mainly core genomes, dispensable genomes and strain-specific genes. The core genome is essential for the basic lifestyle of bacteria, while the dispensable genome provides species diversity, environmental adaptation and other characteristics (Wang et al. 2008). To better understand the pangenome composition of a total of 171,727 genes in the selected isolates, the CDS sequences were clustered. As shown in Fig. 2B, the average number of genes in the core genome was 39.43%, and the proportion of accessory genes was 58.56%, while the proportion of unique genes was 1.8%. Those results indicated that accessory genes were essential for bacterial survival and the basis of their genomic diversity and environmental adaptation. Therefore, the study of auxin genes in S. maltophilia might be able to genetically explain how the organism was altered to adapt to different environments.
Evolutionary analysis of S. maltophilia
To classify the affinities of 42 S. maltophilia isolates, a phylogenetic tree was constructed based on single-copy core genes. As shown in Fig. 3, 42 S. maltophilia isolates were not strictly distributed according to their isolation loci, except that strain 1800 isolated from a contaminated environment as well as strain SJTH1 and WGB211 isolated from shale oil were in an evolutionary branch. Similar consequence was appeared in the mean nucleotide identity clustering heat map of 42 S. maltophilia isolates presented in Fig. 4, wherein the ANI values of strain SJTH1 and WGB211 reached 98.65%, and these three strains 1800 and SJTH1 and WGB211 was 95%, which resulted in their reservation some characteristics from the environment. Combined with previous analyses of the evolutionary relationships of related S. maltobacteria, it could be inferred that isolates from the semblable environmental resources were clustered together, while this conclusion did not apply to clinical isolates due to the differences in the hosts themselves. Our result was consistent with the investigation of S. maltophilia by Yaqian Xiao et al. and the genomic analysis of Aeromonas veronii by Hai-chao Song et al. (Song et al. 2021; Xiao et al. 2021), suggesting that there was no evolutionary correlation between the genome and its ecological niche adaptation.
Functional notes of S. maltophilia
To further investigate their functional properties, COG category and KEGG analysis were performed on the core and non-essential genomes of 19 environmental and 22 clinical strains respectively. COG category of core genes was mainly related to translation, ribosome structure and biogenesis, transcription, amino acid transport and metabolism, and energy production conversion, which were essential for cell growth and/or rapid and efficient response to nutritional environmental sources (Fig. S1). These capabilities conferred a survival advantage to the ever-changing environment (Ying et al. 2019). The accessory genes of environmental and clinical strains were mainly focused on transcription and cell wall /membrane /envelope formation, and there was no significant difference between the proportions of both in the clinical and environmental settings. However, there were significant differences in energy production and conversion as well as auxin transport and metabolism. In addition to the fact that accessory genomic gene activity was closely related to environmental adaptation and environmental tolerance of bacteria, clinical and environmental variation also led to some differences in bacterial function (Bakermans 2018).
Of the core genomic pathways in KEGG, both clinical and environmental isolates were concentrated on metabolism, such as carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins. carbohydrate metabolism, signal transduction, and amino acid metabolism related to some basic life activities of bacteria (Fig. 5). The result suggested that the gene functions of the core genome were mostly conserved in environmental and clinical settings that were related to essential life activities and physiological functions. Extensive signal transduction in the environment allowed the isolates to better respond to changes in the surrounding environment, thus giving them some specific abilities, such as degrading diverse pollutants. The diversity of genes present in different pools indicated that strains selected herein take disparate strategies to adapt to diverse environments (Papon and Stock 2019).
Resistance genotypic diversity within S. maltophilia genomes
The genomes of 22 clinical and 19 environmental isolates were blasted against the database (Table S1) to identify the resistance groups and assesses the distribution of known antibiotic resistance and efflux pump genes in various genomes of S. maltophilia clinical isolates. As presented in Fig. 6, 12 resistance genes were identified in 19 environmental isolates, mainly divided into three major classes of antibiotics resistance-nodulation-cell division (RAD) antibiotic, aminoglycoside 3'-N-acetyltransferase AAC (3'), and aminoglycoside 6'-N-acetyltransferase AAC (6'). The clinical isolates showed a diversity of antibiotic resistance, and 23 antibiotics were identified among the 22 clinical isolates, dividing into 10 categories such as CARB beta-lactamase, L1 family beta-lactamase, Erm 23S ribosomal RNA methyltransferase, major facilitator superfamily (MFS) antibiotic efflux pump, ANT (3'), APH (3'), APH (6') and sulfonamide resistant sul. The greater variety of antibiotic resistance in clinical isolates was mainly due to developing resistance in clinical hosts, and host diversification also showed different resistance to create pressure to treat infections with S. maltophilia. Thus, the selection pressure of the living environment might play a role in the uneven distribution of antibiotic resistance determinants in this context key role (Zhong et al. 2019). On the other hand, environmental strains were found to contain only resistance-nodulation-cell division (RAD) antibiotics efflux pump, while clinical strains contain a major facilitator superfamily (MFS) antibiotic efflux pump except for resistance-nodulation-cell division (RAD) antibiotics efflux pump. This might be due to the intake of foreign antibiotics in the clinical setting, while a high antibiotic environment allowed the efflux pump gene to function as an antibiotic resistance under selective pressure (Dong et al. 2022). Taken together, our results provided extensive genes gain and loss events occurring in S. maltophilia complex genomes produced consistencies in the relationship between the fraction of homologs and evolutionary relatedness, which was likely a crucial factor leading to genetic diversity.