GUO Geng Lin , GAO Hua Sheng , WANG Zhuo Hao , TAN Zhong Ming , HAN Ming Xiao ,CHEN Qi , DU Hong, ZHANG Wei,#, and ZHANG Hai Fang,#

Streptococcus suis(S.suis) is a Gram-positive zoonotic pathogen.S.suisinfection in humans commonly causes meningitis,septicemia,arthritis,and streptococcal toxic shock-like syndrome(STSLS)[1].S.suishas 29 serotypes,of whichS.suisserotype 2 (SS2) has the highest clinical isolation rate and strongest pathogenicity and causes mostS.suishuman infections.About 1,000 different sequence types (STs) were identified inS.suisbased on multilocus sequence typing (MLST).Among these,STs,ST1,and ST7 belong to serotype 2 and are the major hazards ofS.suis[2].In 1998 and 2005,two cases of SS2 human infection outbreak were reported in Jiangsu and Sichuan,China,causing 14 deaths and 38 deaths,respectively[3].

On July 29,2019,a 75-year-old female patient was admitted to The Second Affiliated Hospital of Soochow University for treatment due to “fever accompanied by disturbance of consciousness for one day”.The patient had high leukocyte and calcitonin,severe infection,septic shock,severe condition,and multiple organ damage.The patient was given anti-infection treatment.Unfortunately,the patient died after three days of rescue.Ethical approval was obtained from the ethics committee of Second Affiliated Hospital of Soochow University.

AnS.suisisolate was obtained from the blood sample of this patient and identified by MALDI-TOF MS,16S rRNA,andgdh(a genus-specific gene ofS.suis).Subsequently,acps2I(SS2 specific gene)gene was used to identify this isolate as serotype 2.Furthermore,we found that this isolate belongs to the ST7 type through scanning 7 housekeeping genes used in MLST.Whole genome sequencing is now a powerful tool to study the phylogenetics of bacterial pathogens.Total genomic DNA was extracted and sequenced using the Illumina NovaSeq PE150 platform (Illumina,https://www.illumina.com).This SS2 isolate was named SZ1908,and the whole genome sequence of SZ1908 was deposited in the Genbank database under accession number CP082948.1.SZ1908 contained a single,circular chromosome that is 2,137,458 base pairs (bp) long,with a genomic GC content of 41.29%. The chromosome carries 2029 protein-coding genes,56 tRNA genes,and 12 rRNA genes (Supplementary Figure S1 available in www.besjournal.com).

To explore the genomic differences in this isolate with otherS.suisisolates,all the 33 SS2 isolates obtained from Genbank were retrieved.A whole genome alignment was performed using the software Roary,and the core genes were extracted to build the phylogenetic tree using the Neighborjoining method.Through association analysis with phenotypes such as virulence,sequence type,and isolation source,we found that SZ1908 belongs to the same clade as virulent ST7 SS2.However,it shows some obvious differences from the other human isolates (Figure 1).

To further analyze the genetic difference between SZ1908 and other SS2 isolates,we colinear aligned the genome of SZ1908 with those of three representative SS2 strains,including SC84 (an epidemic ST7 human isolate),P1/7 (an ST1 pig isolate),and T15 (classic avirulent strain).We found that the genomic backbone of SZ1908 is different from that of T15,which has a large fragment transversion and rearrangement in the middle part.The genomic backbone of SZ1908 was much similar to that of the virulent SC84 and P1/7 with only one 100K insert and transversion.However,the genome of SZ1908 is much larger than that of P1/7 and SC84(Figure 2).Also,the SS2 isolates belong to the same tidy clade as SZ1908 (Figure 1).The genomes of these three isolates are highly similar,with only one rearrangement in 600,000 to 700,000 bp.There are differences between single gene deletions and insertions,and through bioinformatic analysis,we found that this is the ICE,which is described below(Supplementary Figure S2 available in www.besjournal.com).

Figure 1.Phylogenic tree of SS2 whole genome sequence based on core gene alignment.The phenotype panel 1 is virulence,panel 2 is STs,and panel 3 is isolation source.The tree scales described the phylogenetic distance.

Figure 2.Colinear Alignment of the genome of SZ1908 with other classic SS2 strains.

More than 100 virulence factors or virulenceassociated genes have now been identified inS.suis.Among these genes is muramidase-released protein(MRP),a cell wall anchored protein with a molecular weight of 136 kD and named MRP because it can be released from the cell wall after treatment of SS2 virulent strain with muramidase.MRP has been traditionally regarded as a virulence marker to identify the virulence ofS.suis[4].Li et al.[5]found that both virulent and avirulent SS2 isolates contain the genemrpalthough there are some differences.The genemrpcould be divided into two parts,a conserved region and a variable region.The variable region is the main factor involved in the virulence of SS2 and the phylogenetic tree constructed based on this region could separate SS2 strins into two clades by virulence[5].In this study,we extracted themrpgene of SZ1908 and those of the other SS2 isolates deposited in Genbank and constructed a phylogenic tree ofmrpgenes.We found that themrpgene sequence of SZ1908 was much closer to those of the classical SS2 avirulent isolates and not in the same clade with those of other human isolates of the SS2 strains (Figure 3).These findings indicate that some other genes could be better markers ofS.suisvirulence.The extracellular factor,suilysin (SLY),also found in the isolate,can be used as a virulence marker ofS.suis.Other potential virulence markers ofS.suisalso found in the isolate include the type I RM system S protein (SSU1589),predicted copper ATPase (SSU0207) proposed by Gottschalk[6],and the major pili subunit SBP2.

Figure 3.Phylogenic tree of mrp sequence of SS2 strains.All human isolate SS2 strains were marked by red,and this isolate was marked by green.

Antimicrobial resistance is one of the most important problems in controlling bacterial diseases.InS.suis,antimicrobial resistance is not that severe.Resistance to tetracyclines,macrolides,and aminoglycosides,especially tetracycline,and erythromycin,is the most prevalent and is associated with resistance genetet(O)anderm(B)[7].Through detecting the minimum inhibitory concentration (MIC) of multiple antimicrobials to SZ1908,we found that SZ1908 is resistant to several antibiotics,including tetracycline and erythromycin(Supplementary Table S1 available in www.besjournal.com). After WGS,the antimicrobial resistance genes (ARGs) were scanned by the online server Resfinder,leading to the identification of seven ARGs,namelymsr(D),erm(B),ant(6)-Ia,

aph(2’)-III,tet(O),tet(40),andmef(A),and the loci of these seven ARGs in the SZ1908 genome are shown in Supplementary Table S2 available in www.besjournal.com.The integrative conjugative element(ICE) is an important mobile genetic element in bacteria that could facilitate the migration of antibiotic resistance determinants[8].Several ICEs have been reported inS.suissince 2012,most of which carried tetracycline and erythromycin resistance genes,which is consistent with the prevalence of antibiotic resistance inS.suis.In SZ1908,a whole length of 189637 bp ICE was identified in the genome,stretching from 561165 to 750802,which is the extra part in SZ1908 without a corresponding part in P1/7 and SC84.Notably,all the seven ARGs were located in this ICE (Supplementary Figure S3 available in www.besjournal.com).Considering that SZ1908 was isolated from a patient,ICE could be an emerging risk to public health.

This study reports a human infection case ofS.suis,which is an important zoonotic pathogen.After genomic analysis,we found that the genome of this isolate is different from those of previous human isolates,and the classicS.suisvirulence markermrpgene could not fit this isolate.Furthermore,this isolate exhibited multidrug resistance and had seven different ARGs located in an integrative conjugative element,which could meditate the migration of antimicrobial resistance.This study has generated information useful for understanding the epidemiology ofS.suisand its implications for public health.

Data AvailabilityThe whole genome sequence of SZ1908 was deposited in the GeneBank database under accession number CP082948.1.

Conflict of InterestsThe authors declare no conflict of interest.

&These authors contributed equally to this work.

#Correspondence should be addressed to ZHANG Hai Fang,E-mail: haifangzhang@suda.edu.cn,haifangzhang@sina.com;ZHANG Wei,E-mail: vszw@njau.edu.cn

Biographical notes of the first authors: GUO Geng Lin,male,born in 1995,PhD Student,majoring in bacteria pathogen pathogenesis and genomics;GAO Hua Sheng,male,born in 1977,majoring in clinical microbiologist.

Received:January 17,2022;

Accepted:April 11,2022