3.1 The phenotype, antimicrobial susceptablities, and biofilm formation
There are five S. maltophilia isolates identified from the 63 samples collected from community in China, named SMYN41, SMYN42, SMYN43, SMYN44, and SMYN45, respectively. Almost all of the five S. maltophilia strains were resistant to AM, GM, PIP, CTX, ATM, IPM, and EM, whereas they are almost sensitive to SXT, SCF, LVX, NOR, CIP, and MIN according to CLSI breakpoints (M100-S23) (Table 1, at the end of this text).
The OD values of biofilm formation of S. maltophilia SMYN41-SMYN45 in OD595 were ranged from 0.101 to 0.218, whereas the ODc is 0.051 (Table 1). The S. maltophilia SMYN41 has produced weak biofilm, while SMYN42, SMYN43 and SMYN45 have had moderate biofilm. And SMYN44 is a strong biofilm-producer.
3.2 Characteristics of the whole genome
The general genomic features of the five isolates sequenced in this study are summarized in the Additional file 1. Whole genome sequences of strain SMYN41, SMYN42, SMYN43, SMYN44, and SMYN45 have total sized ranging from 4,371,193 to 4,897,474 bp with no plasmid. The genomes consist of 72, 18, 17, 62, and 32 contigs with a G+C content 66.60%, 66.72%, 66.31%, 66.72%, and 66.59%, respectively. The predicted genes were then annotated by the COG, KEGG, and GO gene databases (Additional file 2-16). Specifically, a total of 3070 (67.94%), 2828 (72.36%), 2828(72.46%), 2986(70.31%), and 2938(71.78%) genes that were functionally annotated according to GO were classified into 3 categories (biological process, cellular component, and molecular function). A total of 3825 (84.64%), 3419 (87.49%), 3419(87.60%), 3608(84.95%) and 35.47(86.66%) genes belonging to 24 categories were annotated from the COG database. Moreover, based on searches against the KEGG database, 1952(43.20%), 1865(47.72%), 1863(47.73%), 1983(44.57%), and 1886(46.08%) genes were predicted.
3.3 Biofilm-forming relative genes
There are forty genes associated with different mechanisms of biofilm formation in S. maltophilia SMYN41-SMYN45 that annotated based on the NCBI PGAP (Figure 1). All these genes were located on the chromosome. The genes responsible for polysaccharide production (spgM, rmlA, and rmlC) [26,36–38], quorum sensing (QS) (rpfF, ax21, and smoR) [39–45], and flagella (fleQ, flgE/G/G/K/I, flhA, fliF/I/K/M/N/O/A, and fimV) [46–50] were identified in S. maltophilia SMYN41-SMYN45 [18]. Several other biofilm related genes (purE/D/C/I, guaA, and ravS) were annotated in the five strains [46]. Moreover, fliD gene was annotated in SMYN42-SMYN45 [46]. SMYN44 and SMYN45 contain fimbriae gene smf-1[51], polysaccharide production gene xanA[46], and other gene purK [46].
3.4 Antimicrobial resistance analysis
A total of thirty-four genes involved in different mechanisms of drug resistance were annotated and identified based on the NCBI PGAP and CARD (Figure 2). All these genes were located on the chromosome. Several genes encoding RDN family (smeABC, smeDEF, adeF, MntP, and MacB), SMR family (qacJ), and MFS efflux pumps (bcr/CflA, emrCBAsm, and TolC ptotein family) were identified in SMYN41-SMYN45. The five genome sequences also contain a variety of AMR genes, including those conferring aminoglycoside resistance (aph(3’)-II and aph(6)), β-lactam resistance (blaL1 and blaL2), macrolide resistance (MacB) and fluoroquinolone resistance (qnr, gyrA, and parC). The presence of point mutation in gyrA and parC gene may be responsible for fluoroquinolone resistance in strains. Furthermore, SMYN44 and SMYN45 contain smeS, and another aminoglycoside resistance gene aac(3’)-Iz was identified in SMYN41. Notably, aminoglycoside resistance genes (aph(3')-IIc and aac(6')-Iz) as well as β-lactamaseblaL1 were confirmed as acquired resistance genes based on ResFinder4.0 at CGE. However, none of these 5 genomes encoding SXT resistance related genes (sul1, sul2, and dfrA).
The AMR gene profiles of SMYN41-SMYN45 were compared to those of the twenty-five S. maltophilia isolates obtained from the GenBank database (Figure 2). According to the origin of the strains, S. maltophilia SMYN41-SMYN45 conferred similar AMR genes in all human derived strains. SMYN41 shares almost identical antibiotic genes compared to S. maltophilia k279a, and S. maltophilia SMYN44-SMYN45 possessed similar antibiotic genes. Moreover, animal origin isolates contained the fewest AMR genes and did not contain SXT and fluoroquinolone related resistance genes. Environment derived strains contained different kinds of AMR genes and the genes fewer than that in human origin strains. Overall, 28 out of 30 S. maltophilia isolates conferred three or more classes of antibiotic resistant genes. All isolates were harbored sme-related efflux pumps. Most S. maltophilia isolates (25/30) contained SMR family (qacJ or qacG) associated with resistance to disinfecting agents and antiseptics. Almost all strains contained macrolide resistance gene macB except S. maltophilia JV3. β-lactam resistance genes (28/30) and aminoglycoside resistance genes (27/30) were present in most of S. maltophilia isolates. Sm32COP and ZBG7B did not confer the former, as well as the latter were not identified in D457, W18, and ZBG7B. A part of strains (17/30) conferred quinolones resistance genes qnr and gyrA/parC. But no mutations were found in the latter. Only S. maltophilia W18 conferred MFS efflux pumps arlR and norA/C, β-lactamaseblaZ, and SXT resistance gene dfrA, while none of other genomes conferred.
3.5 Phylogenetic analysis
We have downloaded the complete sequences of twenty-five S. maltophilia strains from GenBank and the characteristics are summarized in Table 2. These strains are from human (n = 5), animal (n = 5), or environment (n = 15). The complete genomes of these sequences have total sizes ranging from 4,065,399 to 4,949,420 bp. Most antibiotic phenotypes of these strains are unknown that strains for which the antibiotic resistance profile is not described in the references.
The phylogenetic tree is in Figure 2. Most of strains are not grouped within clusters from the origins. The environmental derived strain CSM2 from Mexican laboratory sink clustered with human strain SMYN41 from China. Human derived strains SMYN42 and SMYN43 clustered with animal origin strains Sm32COP and SmSOFb1, environmental origin isolate mecca, and human isolate SM16975. Moreover, strain k297a from human origin and BurE1 from Burkina environment clustered with SMYN44 and SMYN45 from Chinese human origin and SmF3 from France cattle manure. These confirmed that the phylogeny does not cluster these strains by their geography and origin (human, environmental or animal origin).
In the same way, this phylogeny is not grouped within clusters by the multidrug resistant and antibiotic sensitive strains. In spite of the lack of information for many sequenced strains, according to the known drug-resistant phenotype, for instance, the MDR strain k297a, BurE1, BurA1 and sensitive strain Sm32COP, R551-3, and PierC1 seem to group within different clusters. Although BurE1 is genetically close to k297a, it cannot confirm MDR strains are grouped within clusters. The number of resistance genes or putative resistance genes is not related to resistant phenotype. For example, strain BurE1 are multi-resistant bacteria (Table 2).
3.6 MLST results
Based on the housekeeping genes under the Oxford MLST scheme, we searched and submitted the sequences of these thirty strains on pubMLST (Additional file 17). MLST sequencing showed that S. maltophilia SMYN42, SMYN43 and SMYN45 are new STs, and SMYN41-SMYN45 belong to ST325 (101, 264, 563, 271, 211, 294, and 157 for atpD, gapA, guaA, mutM, nuoD, ppsA, and recA), ST926 (13, 28, 564, 116, 212, 143, and 22), ST926 (13, 28, 564, 116, 212, 143, and 22), ST31 (3, 4, 24, 7, 7, 22, and 7), and ST928 (1, 1, 326, 3, 25, 295, and 1), respectively. Isolates SMYN42 and SMYN43 possess the same housekeeping gene alleles and both belong to ST926. Among these twenty-five sequences downloaded from GenBank, fourteen types are the new STs (ST929-ST942). Eight isolates consisted of existing types in the database without any duplicate sequence types. And we could not find complete trusted alleles or hits in the other five isolates, including Sm46PAILV, SmSOFb1, W18, and ZBG7B. The MLST minimum spanning tree has further confirmed that this phylogeny is not grouped within clusters by the source or antibiotic resistance (Figure 3).