IS26-Mediated Genetic Rearrangements in Salmonella Genomic Island 1 of Proteus mirabilis

Salmonella genomic island 1 (SGI1) is an integrative mobilizable element integrated into the chromosome of bacteria, which plays an important role in the dissemination of antimicrobial resistance genes. Lots of SGI1 variants are found mainly in Salmonella enterica and Proteus mirabilis. In this study, a total of 157 S. enterica and 132 P. mirabilis strains were collected from food-producing animals in Sichuan Province of China between December 2016 and November 2017. Detection of the SGI1 integrase gene showed that three S. enterica and five P. mirabilis strains were positive for SGI1, which displayed different multidrug resistance profiles. Five different SGI1 variants, including two novel variants (SGI1-PmBC1123 and SGI1-PmSC1111), were characterized by whole genome sequencing and PCR linkage. In two novel SGI1 variants, IS26-mediated rearrangements resulted in large sequence inversions of the MDR regions extending outside the SGI1 backbone. The sul3-type III class 1 integron (5′CS-sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) and gene cassettes aac(6′)-Ib-cr-blaOXA–1-catB3-arr-3 are found in SGI1-PmSC1111. Mobilization experiments indicated that three known variants were conjugally mobilized in trans to Escherichia coli with the help of a conjugative IncC plasmid. However, the two novel variants seemed to lose the mobilization, which might result from the sequence inversion of partial SGI1 backbone. The identification of the two novel SGI1 variants in this study suggested that IS26-mediated rearrangements promote the diversity of SGI1.


INTRODUCTION
Genomic islands (GIs), such as integrative and conjugative elements (ICEs) and integrative mobilizable elements (IMEs), are distinct regions integrated into the chromosome of bacteria and acquired via horizontal transfer (Bellanger et al., 2014;Partridge et al., 2018). GIs often contain various genes endowing theirs hosts with new traits, like antimicrobial resistance and virulence that enhance bacterial adaptation to environment (Bellanger et al., 2014). Salmonella genomic island 1 (SGI1) is an IME initially identified in the multidrug resistance (MDR) Salmonella Typhimurium DT104 clone (Boyd et al., 2001). SGI1 (42.4 kb) is comprised of a backbone containing 28 ORFs (S001-S027 and S044) and a 13 kb MDR region that consists of a complex In4-type class 1 integron named In104 (Boyd et al., 2001;Levings et al., 2005). It can form extrachromosomal circular form and is specifically mobilized in trans by conjugative IncA/C plasmids (Doublet et al., 2005;Douard et al., 2010). In recent years, the mobilization mechanism of SGI1 with the help of IncA/C plasmids has been revealed in some studies and many aspects have been explored including the basic transfer elements (Carraro et al., 2014;Kiss et al., 2015Kiss et al., , 2019Siebor et al., 2016). SGI1 was reported in Proteus mirabilis in 2007 (Ahmed et al., 2007), and recently found in Morganella morganii, Providencia stuartii and Escherichia coli (Schultz et al., 2017a;Cummins et al., 2019;Soliman et al., 2019), indicating that SGI1 has a broad host bacterial range and has the potential to spread among enterobacteria.
In the present study, we characterized the SGI/PGI genomic islands in Salmonella enterica and P. mirabilis of food-producing animal origin in Sichuan Province of China and described two novel SGI1 variants in P. mirabilis.

Bacterial Strains and Detection of SGI1 and Relative Islands
A total of 157 S. enterica strains (61 from swine and 96 from chicken) and 132 P. mirabilis strains (74 from swine and 58 from chicken) isolated from diseased tissues or anal swabs of animals among 30 poultry and 30 swine farms in Sichuan Province between December 2016 and November 2017. All isolates were identified using an automated system (BD Diagnostic Systems, Sparks, MD, United States). The presence of SGI/PGI/AGI/GIPmi1 was screened by PCR targeting the integrase gene (the primers used to detection are listed in Supplementary Table S2) (Schultz et al., 2017b). Positive PCR products were sent to Chengdu Qingke Biological Engineering Technology & Services Co., Ltd., and sequenced by ABI 3730xl DNA Sequencer (Applied Biosystems, United States).

Whole Genome Sequencing and Analysis
All SGI/PGI-positive strains were sequenced using Illumina HiSeq platform (400-bp paired-end reads with about 200-fold average coverage). The draft genomes were assembled using software SPAdes_3.12.0. The gaps among contigs that carried SGI1 fragments were filled in by PCR linkage. Because the complete genetic structures of SGI1 in strains PmBC1123 and PmSC1111 could not be assembled by PCR linkage, whole genomes of those two strains were further sequenced using PacBio RS II sequencing instrument (100-fold average read depth). The chromosomes were assembled into one scaffold using software SMRT portal v.3.2.0. The MDR regions were confirmed by PCR linkage between regions belonging to non-repeated genetic elements. Multi-locus sequence type of S. enterica and acquired antimicrobial resistance genes were identified by MLST 2.0 1 and ResFinder 3.1 2 , respectively. The complete nucleotide sequences of SGI1 variants were analyzed using the BLAST programs 3 . SNPs from genomes of the strains positive for SGI/PGI were called and a phylogeny based on the concatenated alignment of the high quality SNPs was inferred using CSI Phylogeny 1.4 4 with parameters as defaults.

Mobilization Assays of SGI1
Many SGI1 variants can form the circular extrachromosomal forms that are conjugally mobilized in trans to other bacteria with the help of the conjugative IncA/C plasmid (Hall, 2010). The circular extrachromosomal forms of SGI1 variants were detected through two rounds of PCR amplification using primers listed in Supplementary Table S2. Mobilization assays were carried out as previously described (Siebor et al., 2016), using E. coli C600 harboring an IncC plasmid pR55 as recipient strain. Transconjugants were selected on Shigella and Salmonella agar plates containing 300 mg/L rifampicin and trimethoprim (30 mg/L)/streptomycin (30 mg/L). The transfer frequency of SGI1was determined by dividing the number of E. coli SGI1 transconjugants by the number of P. mirabilis or S. enterica donor cells (Douard et al., 2010). The transconjugants were further  examined for the presence of SGI1 integrase gene and the location of SGI1 in E. coli with primers listed in Supplementary Table S2.

RESULTS AND DISCUSSION
Prevalence of SGI/PGI in S. enterica and P. mirabilis Detection and sequence analysis of SGI/PGI/AGI/GIPmi1 integrase gene showed that 3 S. enterica and 5 P. mirabilis strains were positive for SGI1. Antimicrobial susceptibility testing indicated those 8 strains displayed different MDR profiles ( Table 1) (P. mirabilis is intrinsically resistant to doxycycline and polymyxin B). S. Albany strain SCMYP1 of swine origin exhibited resistance to the third generation cephalosporins and polymyxin B. Two P. mirabilis strains (PmSN55 and PmDJ107) of chicken origin were resistance to amoxicillin-clavulanic acid, cefoxitin and the third generation cephalosporins. Three SGI1-containg S. enterica strains belonged to different STs (Table 1). A total of 28,848 SNPs were called and phylogenetic analysis showed the five P. mirabilis strains were not clonally related.

Mobilization of SGI1
A free circle can be formed after excision of SGI1 from the chromosome (Doublet et al., 2005). SGI1 appeared to be nonself-transmissible, but it could potentially be integrated into the chromosome of another bacterial species by the help of IncA/C plasmid (Douard et al., 2010;Siebor et al., 2016). In the recipient strain, the circular form of SGI1 integrates in a specific site at the 3 end of the chromosomal trmE gene (Doublet et al., 2005). The circular forms of SGI1 in all strains except for PmBC1123 and PmSC1111 were detected by two rounds of PCR amplification. We did not detect the circular form of SGI1-PmBC1123 because of the inversion of the right direct repeat in SGI1-PmBC1123. The circular form of SGI1-PmSC1111 could not be detected through three independent experiments. Mobilization assays showed that the three known SGI1 variants (SGI1-F, SGI1-I and SGI1-W) in S. enterica or P. mirabilis could be conjugally mobilized to E. coli and was incorporated into the 3 -end of trmE. The conjugative transfer of them were detected at frequencies between 10 −6 and 10 −7 , suggesting that these SGI1s can be transferred between bacterial species (Table 2). However, the mobilization of SGI1-PmBC1123 and SGI1-PmSC1111 failed despite three independent attempts. The results indicate that the sequence inversion of partial SGI1 backbone may result in the loss of mobility of SGI1. We supposed that the inversion of the right direct repeat in SGI1-PmBC1123 may lead to losing the capability to form a circular form and then mobility. Nevertheless, in SGI1-PmSC1111, IS26mediated rearrangements resulted in inversions of the backbone was supposed to affect the expression of some genes related to mobilization. The mechanism needs further study to clarify.

Other Resistance Genes That Were Not Associated With SGI1
The acquired antimicrobial resistance genes in SGI1-containing S. enterica and P. mirabilis isolates are listed in Table 3. S. Albany strain SCMYP1 harbors bla CTX−M−55 and mcr-1, explaining the resistance to third generation cephalosporins and polymyxin B, respectively. P. mirabilis strains PmSN55 and PmDJ107 harbors AmpC cephalosporinase gene bla CMY−2 carried by an 11.7-kb contig that is identical to the corresponding region of SXT/R391 ICE ICEPmiJpn1 (Harada et al., 2010;Lei et al., 2016), indicating the bla CMY−2 gene in those two strains might be carried by ICEPmiJpn1. It is notable that strain PmSC1111 harbors the multiresistance gene cfr that is also carried by SXT/R391 ICE (accession no. CP034090). Very recently we reported a novel SXT/R391 ICE that carried cfr, bla CTX−M−65 , fosA3, and aac(6 )-Ib-cr in P. mirabilis and could be transferred to E. coli (Lei et al., 2018a). Taken together, SXT/R391 ICE could mediate the dissemination of clinically important resistance genes in P. mirabilis, which needs to draw more attention.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the GeneBank, MH998664, MH998665, CP034091 and CP034090.

ETHICS STATEMENT
This study was carried out in accordance with the recommendation of ethical guidelines of Sichuan University. The protocol was approved by the Sichuan University Animal Ethics Committee. Individual informed consent for the use of samples was obtained from all the animal owners.
AUTHOR CONTRIBUTIONS X-CW, C-WL, Z-ZK, and YZ performed the experiments. C-WL analyzed the data and conceived of the study. X-CW, C-WL, and Z-ZK wrote the manuscript. All authors contributed to manuscript revision and approved the final manuscript.