Comparative Analysis of Streptococcus Agalactiae S03 and S07 Isolated From Schizothorax Prenanti With Different Antibiotic Resistance and Virulence

Streptococcus agalactiae (GBS), is an important Gram-positive pathogen of ﬁ sh aquaculture worldwide. In this study, we performed a comparative analysis of GBS S03 and S07 isolated from Schizothorax prenanti and explored the association between phenotypic antibiotic resistance as well as virulence and the genomic characteristics. Antimicrobial sensitivity tests on 12 common antibiotics using the disc diffusion method revealed that the S03 showed resistance to seven antibiotics, while S07 showed sensitivity to the tested antibiotics. Pathogenicity analysis demonstrated greater virulence of S07 than S03. Then, the occurrence of antibiotic resistance and virulence genes was identi�ed using whole-genome sequence (WGS) of S03 and S07. There were mefE, tetO, lnuB, lsaE, APH3', and sat-4 resistance genes present only in S03 genome. And just S03 had gyrA and parC genes mutations. In addition to 51 virulence genes in both S03 and S07 genomes, S07 additionally carried virulence genes associated with invasion, such as SAN_1519, rfbA and cylE genes. There was complete concordance between genotypic evidence and phenotypic characteristics. Virulence factors and phylogenetic analysis showed that S03 and human sources shared an extremely close evolutionary relationship. Our �ndings provide important proof for using WGS as an effective tool of phenotypic predictions of GBS.

WGS analysis and phenotypic characteristics has not been reported.Here, this is the rst report on draft genomes of GBS S03 and S07 isolated from Schizothorax prenanti with different pathogenicity and drugresistance.Draft genomes of GBS S03 and S07 are important additions to the GenBank database of piscine GBS.
In the current study, antimicrobial susceptibility testing, experimental infection and WGS analysis were carried out to compare two GBS clinical strains isolated from Schizothorax prenanti.Phylogenetic analysis was performed to identify their evolutionary relationships in GBS.We attempted to assess the use of WGS to evaluate the genomic diversity and genotypic prediction of drug-resistant phenotype and pathogenicity of them.

Bacterial strains
GBS S03 and S07 were isolated from diseased Schizothorax prenanti, with characteristic clinical and pathogenic meningoencephalitis from the culture collection of the College of Veterinary Medicine, Sichuan Agricultural University, China.Serotyping assigned GBS S03 to serotype III and S07 to serotype Ia by cps cluster.

Pathogenicity experiments
To con rm the pathogenicity and virulence of GBS S03 and S07, we injected the two strain suspensions into healthy Schizothorax prenanti and Danio rerio, intraperitoneally.Healthy Schizothorax prenanti (11.5 ± 1.2 g) and Danio rerio (0.4 ± 0.05 g) were purchased from Sichuan Ya-sh Company and Model Animal Research Center Of Nanjing University, respectively.260 Schizothorax prenanti and Danio rerio were kept in 13 groups equally, respectively, in 120-L plastic tanks (n = 20/tank or group) with aeration and were acclimatized at 24 ± 1°C for 7 days.The Schizothorax prenanti of 6 experiment groups were challenged with 0.1 ml of bacterial suspensions of the S03 and the other 6 groups were injected with the same volume of S07 suspensions in sterile 0.8% NaCl solution at concentrations of 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 cfu•ml − 1 .The control sh (n = 20) were injected with 0.1 ml 0.8% NaCl solution.Similarly, the Danio rerio of test groups were injected intraperitoneally with 0.05 mL of bacterial suspensions of the S03 and S07 in sterile 0.8% NaCl solution at different concentrations mentioned above.The control animals were injected with 0.05 mL 0.8% NaCl solution.Mortality was recorded daily for 21 d and mean lethal dose (LD 50 ) values were calculated.All the sh were euthanized by using 300 mg•L − 1 MS222 (yuanye Bio.Co. Ltd., Shanghai, China) for 21 d.

Whole genome sequencing and annotation
To explore further differences between S03 and S07, their draft genomes were sequenced and analyzed at whole genome level.The genomic DNA of the two strains were extracted using the TIANamp bacteria DNA kit (Tiangen, Beijing, China) and applied for library preparation using the Nextera DNA sample preparation kit (Illumina, San Diego, USA).The samples were accessed for the genome sequencing on an Illumina HiSeqTM 2000 (Illumina Inc., San Diego, USA) using a paired-end 2×100 bp protocol.The whole genome sequences were assembled using CLC Genomics Workbench 10.0 software (QIAGEN, Hilden, Germany).Contigs were initially annotated using Rapid Annotations using Subsystems Technology (RAST) and then manually checked.Resistance-related genes of the two strains were analyzed using ResFinder 2.1 server (Zankari et al. 2012).Gene clusters were classi ed into COG (Cluster of Orthologous Groups of proteins) categories (http://www.ncbinlm.nih.gov/COG/)according to BLAST annotations.

Identi cation of antibiotic resistance genes and virulence factors
To nd antibiotic-resistant genes in two GBS strains genomes, known antibiotic resistance genes were downloaded from the Comprehensive Antibiotic Resistance Database (CARD) and aligned with all coding sequences (CDSs) of those genomes using BLAST (https://card.mcmaster.ca/).A similar method has been applied to identify virulence genes from the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/).

Phylogenetic tree
A phylogenetic tree of GBS strains was constructed based on seven housekeeping genes (adhP pheS atr glnA sdhA glcK and tkt) as previously described (Jones et al. 2003).The alignments of these genes were concatenated into a single sequence alignment and a maximum likelihood tree was reconstructed using MEGA (version 10.0).The allele number and sequence type (ST) were assigned by multilocus sequence typing (MLST) website (https://pubmlst.org/organisms/streptococcus-agalactiae).

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 classi cation 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).The genome sequences of S03 and S07 have been deposited in the NCBI GenBank database under the accession numbers NAPX00000000 and NHZY00000000, respectively.

Antibiotic resistance pro le and antibiotics resistance genes
Antimicrobial susceptibility distribution of S03 and S07 was given in Fig. 1a.The results showed that there were signi cant 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.
Fifty-one virulence genes were identi ed in the both genomes (

Phylogenetic analysis
By MLST, S03 was determined to be ST19, while S07 was ST891.To investigate the phylogenetic relationships of our two isolates to other GBS strains, we used the maximum likelihood method to construct a phylogenetic tree (Fig. 3).Phylogenetic analysis was performed using 38 reported GBS strains.Interestingly, S03 was closely related to Sag158, H002 and SG-M25 strains isolated from human while S07 was grouped separately.

Discussion
Since 2008, GBS has been present and emerging in tilapia in China (Zhang et al. 2008).Liu et al. (2013) reported the rst complete genome sequence of the piscine GBS strain isolated from cultured tilapia in China.In this study, draft genomes of GBS S03 and S07 isolated from Schizothorax prenanti have been recorded.The genome size of S07 was approximately 1.79 Mbp and approximately 0.37 Mbp smaller than S03.There were more predicted CDSs in the S03 genome than in S07.Genome size is the result of a balance between ampli cation and loss of DNA, which evolves toward an optimal state for organismal tness (Devos et al. 2002;Lynch 2006).Furthermore, genomic changes leading to streamlined genomes with increased pathogenicity and virulence are important (Siguier et al. 2014).They might involve fragment recombination and contribute to the different virulence of S03 and S07.
Antimicrobial tests revealed that the GBS S03, showed multidrug resistance, but S07, showed drugsensitive to the tested antibiotics.The differences in drug resistance between the two strains were signi cant, which may have been associated with environmental variability and/or the frequent and abusive use of chemotherapy in aquaculture (Abuseliana et al. 2010; Florindo et al. 2010).In the current study, the phenotypes of two strains were in concordance with genomic characteristics revealed by WGS.Two strains had uoroquinolone genes: parC, gyrA, gyrB, norA and norB, however, just S03 had chromosomal gyrA [81:S-L] and parC [79:S-Y] mutations, explaining high-level uoroquinolone resistance (Kawamura et al. 2003;Salma et al. 2013;Doumith et al. 2017).In addition to two macrolide resistant genes (macB and ermB) and one tetracycline resistant gene tetM, S03 additionally had mefE and tetO, which contributed to high-level macrolide and tetracycline resistance (Poyart et al. 2003;Doumith et al. 2017), as observed in this study.These results are consistent with what we have previously found through traditional methods (Deng et al. 2019), but WGS is clearly more e cient and accurate.Meanwhile, GBS S03 had additional lnuB, lsaE, APH3', and sat-4 genes not present in S07 strain, the rst two of which play an important regulatory role in lincosamide resistance (Faccone et al. 2010).The combination of APH3' and sat-4, associated with transposon Tn5405 among Gram-positive bacteria, encodes resistance to aminoglycosides (except gentamicin) and streptothricins (Derbise et al. 1997;Werner et al. 2003).These results reveal a trend towards concordance between resistance phenotype and genotypic evidence for antibiotic resistance.
The pathogenicity of the two strains were compared by intraperitoneal injection in Schizothorax prenanti or Danio rerio, which demonstrated that S07 showed much greater virulence than S03.S07 also carried more virulence genes associated with invasion, such as SAN_1519, rfbA and cylE genes.However, it was counter to the previous report on GBS from Tilapia in Thailand (Areechon et al. 2016), which is most likely related to the diversity in host, area and strain.Fifty one virulence genes in both GBS S03 and S07 involved in attachment, persistence, evading host defenses, tissue penetration, and toxin-mediated diseases had been identi ed in the GBS genomes, providing evidence of the pathogenicity of GBS (Godoy et al. 2013;Chen et al. 2014;Kayansamruaj et al. 2014).Differences in virulence genes between the two strains are mainly re ected in the genes encoding pili, including SAG_1404-1408, gbs0628-0629 and SAN_1519.Genes encoding pili in GBS are located within two distinct loci, denoted pilus islands 1 and 2 (PI-1 and PI-2), and comparative analysis of available genomes revealed two variants of PI-2, designated PI-2a and PI-2b (Rosini et al. 2006).Gbs0628-0629 genes, SAG_1404-1408 genes, and SAN_1519 gene are located in PI-1, PI-2a and PI-2b, respectively (Rosini et al. 2006;Nobbs et al. 2008).Pili are supposed to be important virulence factors (Nobbs et al. 2008).In GBS, pilus 1 is related to the immune escape, pilus 2a confers formation of bio lms, and pilus 2b is linked to the ability of attachment and invasion (Rinaudo et al. 2010;Jiang et al. 2012).In this study, S03 carried PI-1 plus PI-2a type pilus islands while S07 carried PI-2b type pilus island, which suggested there was concordance between pathogenicity and WGS result.Previous studies showed that most of the sh-sources strains carried PI-2a type, PI-2b type or PI-1 plus PI-2b type pilus islands, while in human strains, a great number of GBS carried PI-1 plus PI-2a type or PI-1 plus PI-2b type (He et al. 2017).
Interestingly, phylogenetic analysis showed that S03 was closely related to human sources (Sag158, H002 and SG-M25) while S07 was grouped separately.Combined with genome size and phenotypic characteristics, we hypothesized that they had different origins and evolutionary directions.Further, bestowed pili types and phylogenetic analysis, we hypothesized that S03 had a common source with human GBS or the possibility of cross-infection with human strains.Unfortunately, we currently do not have adequate epidemiological information to con rm this hypothesis.Although the knowledge about the ability of GBS to cross the interspecies barrier and allow human-derived strains to infect animals, or vice versa, is poorly understood, Pereira et al. (2010) demonstrated that GBS strains from human and bovine origins could infect sh.Rajendram et al. (2016) found that human could be infected by shisolated GBS by consuming of raw sh.Therefore, we should attach great importance to cross-host transmission of GBS and its potential threat to public health security.In addition, whether our two strains in this study will exchange and transfer resistance genes and virulence genes in nature, with the appearance of new strains with great toxicity and extreme drug resistance, which deserve further research and attention.

Conclusions
In this study, GBS S03 and S07 isolated from Schizothorax prenanti showed greatly distinct phenotypic characteristics, including drug-resistant phenotype and pathogenicity, and they were diverse at the genome level.There was nished concordance between genotypic evidence and phenotypic characteristics.Our ndings provide important proof for using WGS as an operational tool of phenotypic predictions of GBS.survival curves of sh injected with S07 suspensions at 1.0×109, 1.0×108, 1.0×107, 1.0×106, 1.0×105,

Figures
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Figure 1 Heat
Figure 1

Table 1
General features of GBS S03 and S07.