| Glaesserella parasuis is a respiratory tract pathogen characterized by fibrinous polyserositis,pleurisy,polyarthritis and meningitis,and it is considered to be one of the most serious pathogenic microorganisms in the pig industry.Antibiotics are effective means of clinical prevention and treatment to this pathogen.With the irrational use of antibacterial drugs,sensitivity of G.parasuis to commonly used antimicrobial drugs was reduced.The resistance to different drugs were conferred by the expression of ARGs,and the spread of ARGs promotes the spread of drug resistance.As the carriers of ARGs transmission,mobile genetic elements(plasmids,transposons,integrons and integrative and conjugative elements)play an important role in the transmission of drug resistance.In this study,the susceptibility test and detection of ARGs in 211 isolates were helpful to screen potential mobile genetic elements.The characteristics of plasmids,transposons and integrative and conjugative elements carrying ARGs prevalent in G.parasuis were analyzed,which laid a foundation for the ARGs transmission mechanism.The genetic evolution relationship of the strains was elaborated through serotype identification and MLST analysis,which provided the basis for clinical establishment of drug resistance database and the reference guidance for prevention and control of the spread of drug resistance in G.parasuis.The MICs of the 211 strains were determinated,and the MIC90 of oxytetracycline,amoxicillin,streptomycin,tylosin and sulfamethoxazole/trimethoprim in the selected antibacterial agents showed high MICs of 64,16,128,16 and 32 μg/m L,respectively.Then the whole genomes of 211 isolates were sequenced using Illumina Hi Seq X Ten,and ARGs were detected using Res Finder database in CGE server.A total of 17 ARGs were detected in 211 isolates,and the most popular genes were tet(B),bla Rob-1,str A and sul2,with the detection rates with 13.7%、9.5%、9.0% and 9.0%.In addition,a novel β-lactamase Bla Rob-3 was identified.Compared with the amino acid sequences of the Bla Rob-1,two amino acids leucine(L)and threonine(T)were added at the 16 th and17th positions of Bla Rob-3.Bioinformatics analysis showed that 17 strains(8.1%,17/211)of 211 isolates carried drug-resistant plasmids.And four plasmid types: Type I(mob C,mob A and ori V)、Type II(mob C-like,mob A-like,orf5 and rep)、Type III(mob C,mob A/L and rep)and Type IV(rep)were identified.Among them,type IV plasmids(4.3%,9/211)were the most prevalent plasmids,followed by type I(2.8%,6/211),and type II(1.9%,4/211).Type III plasmids were not detected in this study.None of these plasmids can be transferred by conjugation test,but they can be transmitted within and across species through transformation.Moreover,eight of the 211 isolates carried MTn Sag1 transposons containing lnu(C)gene,but none of these transposons could self-transfer.It was found that MTn Sag1 can be inserted into the chromosome of serotype 7 G.parasuis for the first time.It is the first time that MTn Sag1 has been located in the chromosome of serotype 7 G.parasuis isolates.By comparing the amino acid sequences of 66 Lnu(C)carrying MTn Sag1 transposons in this study and NCBI database,eight variants of the lnu(C)genes: lnu(CI)-lnu(CVIII)were found.lnu(CIII)was the most popular type,and the lnu(C)genes of the eight isolates in this study belonged to lnu(CIII)and lnu(CV).The whole genomes of six ICE-carrying strains YHP170504,EHP1804,YHP1818,GHP1808 GHP1807 and YHP1815 were subsequently sequenced by Illumina Nextseq 500 and Oxford Nanopore Technologies Min ION.To our knowledge,this is the first time a ICE carrying seven ARGs [bla Rob-3,tet(B),aph A1,str A,str B,aac(6)’-Ie-aph(2’)-Ia and sul2] and five variants with different ARGs profiles have been reported in G.parasuis,and a complete set of replication and type IV secretion system were identified.Moreover,two novel insertion sequences,ISGpa1 and ISGpa2,were identified in G.parasuis for the first time.The serotypes and STs of 211 isolates were determined by PCR.Serotype analysis showed serovar 4,5/12 and 13 were the most prevalent serovars with the rates of 30.8%,20.9% and 13.3%,eight isolates were classified as untypeable(3.8%),and serovar 3,6 and 9 were not detected.MLST typing showed that all 211 isolates could be typed,but the distribution of ST types was not concentrated,distributed in 96 ST types.ST176(9 strains),ST449(9 strains)and ST489(9 strains)were the most popular STs among all isolates,followed by ST280(6 strains)and ST519(6 strains),and other ST types were 1-5 strains,respectively.In addition,30 alleles were found and 67 new ST types were identified in this study.In conclusion,through whole-genome sequencing and bioinformatics analysis,we analyzed the prevalence and distribution of ARGs in 211 isolates,described the molecular characteristics of four types plasmids and found that MTn Sag1 can be inserted into the chromosome of serotype 7 G.parasuis for the first time.In addition,a novel integrative and conjugative element ICEHpa1 and five ICEHpa1 variants carrying different ARGs were identified in this study.These findings revealed the transmission mechanism of different ARGs in G.parasuis. |
