3.1. General features of the GBS S03 and S07 genomes
Characteristics of the two genomes are summarized in Table 1.The circular maps of two strains showed that the genome of S03 was 2,156,979 bp in length (Fig. S1a), while that of S07 was 1,790,578 bp in length (Fig. S1b). Genome sequence of both strains showed 35% G + C contents. RAST server predicted the 2,160 and 1,641 CDSs for S03 and S07 strains, respectively. On the basis of COG classification of S03 and S07, 23.7% and 22.6% of CDSs were responsible for information storage and processing, whereas 37.5% and 39.4% were for metabolism (Fig. S2).
Table 1
General features of GBS S03 and S07.
Features
|
S03
|
S07
|
Host
|
Schizothorax prenanti
|
Schizothorax prenanti
|
Sequence type
|
ST19
|
ST891
|
Serotyping
|
III
|
Ia
|
Genome size (bp)
|
2,156,979
|
1,790,578
|
G + C content (%)
|
35
|
35
|
Total CDSs
|
2,160
|
1,641
|
CDSs responsible for information storage and processing (%)
|
23.7
|
22.6
|
CDSs responsible for metabolism (%)
|
37.5
|
39.4
|
Transfer RNA
|
42
|
29
|
The genome sequences of S03 and S07 have been deposited in the NCBI GenBank database under the accession numbers NAPX00000000 and NHZY00000000, respectively.
3.2. Antibiotic resistance profile and antibiotics resistance genes
Antimicrobial susceptibility distribution of S03 and S07 was given in Fig. 1a. The results showed that there were significant differences in resistance between the two strains. Antimicrobial tests revealed that S03 showed multidrug resistance against OFX, NOR, ENR, TET, DOX, ERY and CLIN (Table S1). By contrast, S07 was sensitive or intermediate to all 12 tested antibiotics.
The genomes of GBS S03 and S07 showed that S03 had 20 resistance genes, while S07 had 14 resistance genes (Fig. 1b and Table S2). In addition to two macrolide resistant genes (macB and ermB) and one tetracycline resistant gene tetM, S03 also had mefE and tetO genes. Meanwhile, S03 had additionally lnuB, lsaE, APH3', and sat-4 genes not present in S07. Both strains carried genes encoding resistance to fluoroquinolone: parC, gyrA, gyrB, norA and norB, however, just S03 had chromosomal gyrA [81:S-L] and parC [79:S-Y] mutations (Fig. S3).
3.3. Pathogenicity experiments and virulence genes
At 21 days post-infection (dpi), GBS S07 induced higher levels of mortality and revealed a greater virulence than S03 in the pathogenicity experiments (Fig. 2). Concretely, The LD50 value of S03 for Schizothorax prenanti and Danio rerio was 5.27×108 cfu·ml− 1 and 7.09×107 cfu·ml− 1 respectively, while that of S07 was 8.9×103 cfu·ml− 1 for Schizothorax prenanti and 1.88×103 cfu·ml− 1 for Danio rerio. S03 strain caused 25% and 60% mortality in Schizothorax prenanti at the 1.0×108 and 1.0×109 cfu·ml− 1, while S07 caused 55%, 75%, 95%, 100%, 100% and 100% at 1.0×104, 1.0×105, 1.0×106, 1.0×107, 1.0×108 and 1.0×109 cfu·ml− 1 concentrations respectively. Danio rerio had 20%, 35%, 55% and 70% cumulative mortality caused by S03 at 1.0×106, 1.0×107, 1.0×108, 1.0×109 cfu·ml− 1, while caused by S07 were 70%, 85%, 95%, 100%, 100% and 100% at 1.0×104, 1.0×105, 1.0×106, 1.0×107, 1.0×108, 1.0×109 cfu·ml− 1 respectively. There was no mortality in the control groups during the observation period.
Fifty-one virulence genes were identified in the both genomes (Table 2). Among these, bibA, dltA, fbsA, pavA, psaA, srtA, fbsB, pavB, lap, lep, lmb and srr-1 were involved in attachment; CLL_A 2400, hylB, lgt, mf3, and sugC were involved in invasion; cfb, cpsA-L, cpsA-Y, manA, neuA-D, uppS, rgpA, rmlA, scpA, rgpB, scpB, oppF, rgpG, sip, BC5263, EFD32, EFD32_0765 and SMU.322c were involved in evading/destroying host defenses; and other virulence genes, included were coxK2, clpC, clpE, clpP, cppA, msbA, nanA, gbpB, fhuC, licD, lisR, lplA1, groEL, htrA/deg, CT396 and stp. Comparison of the virulence genes between S03 and S07 showed differences. S03 carried SAG1404-1408, rib, SAK_0778–0779 and gbs0628-0629 associated with attachment genes, while S07 were carrying SAN_1519, rfbA and cylE involved in invasion genes.
Table 2
Virulence genes of GBS S03 and S07.
Class
|
S03
|
S07
|
Attachment
|
bibA, dltA, fbsA, pavA, psaA, srtA, fbsB, pavB, lap, lep, lmb, srr-1, rib, SAG1404, SAG1405, SAG1406, SAG1407, SAG1408, SAK_0778, SAK_0779, gbs0628, gbs0629
|
bibA, dltA, fbsA, pavA, psaA, srtA, fbsB, pavB, lap, lep, lmb, srr-1
|
Invasion
|
CLL_A 2400, hylB, lgt, mf3, sugC
|
CLL_A 2400, hylB, lgt, mf3, sugC, SAN_1519, rfbA, cylE
|
Evading/destroying host defenses
|
Cfb, cpsA-L, cpsA-Y, manA, neuA-D, uppS, rgpA, rmlA, scpA, rgpB, scpB, oppF, rgpG, sip, BC5263, EFD32, EFD32_0765, SMU.322c
|
Cfb, cpsA-L, cpsA-Y, manA, neuA-D, uppS, rgpA, rmlA, scpA, rgpB, scpB, oppF, rgpG, sip, BC5263, EFD32, EFD32_0765, SMU.322c
|
Other virulence genes
|
coxK2, clpC, clpE, clpP, cppA, msbA, nanA, gbpB, fhuC, licD, lisR, lplA1, groEL, htrA/deg, CT396, stp
|
coxK2, clpC, clpE, clpP, cppA, msbA, nanA, gbpB, fhuC, licD, lisR, lplA1, groEL, htrA/deg, CT396, stp
|