Emergence of a Hybrid IncI1-Iα Plasmid-Encoded blaCTX-M-101 Conferring Resistance to Cephalosporins in Salmonella enterica Serovar Enteritidis

The increasing resistance to cephalosporins in Salmonella poses a serious threat to public health. In our previous study, the blaCTX-M-101 gene, a new blaCTX-M variant, was first reported in Salmonella enterica serovar Enteritidis (S. Enteritidis). Here, we further analyzed the genome characterization, transferability, and resistance mechanism of one S. Enteritidis isolate (SJTUF14523) carrying blaCTX-M-101 from an outpatient in 2016 in Xinjiang, China. This strain was a multidrug resistance (MDR) isolate and exhibited resistance to ceftazidime (MIC = 64 μg/mL), cefotaxime (MIC = 256 μg/mL), and cefepime (MIC = 16 μg/mL). Phylogenetic analysis revealed that SJTUF14523 had a close relationship to another S. Enteritidis isolate from the United States. In the presence of plasmid p14523A, there were 8- and 2133-fold increases in the MICs of cephalosporins in Escherichia coli C600 in the conjugation. Gene cloning results indicated that blaCTX-M-101 was the decisive mechanism leading to ceftazidime and cefotaxime resistance that could make the MICs break through the resistance breakpoint. Plasmid sequencing revealed that the blaCTX-M-101 gene was located on an IncI1-Iα transferable plasmid (p14523A) that was 85,862 bp in length. Sequence comparison showed that p14523A was a novel hybrid plasmid that might have resulted from the interaction between a homologous region. Furthermore, we found a composite transposon unit composed of ISEcp1, blaCTX-M-101, and orf477 in p14523A. ISEcp1-mediated transposition was likely to play a key role in the horizontal transfer of blaCTX-M-101 among plasmids in S. Enteritidis. Collectively, these findings underline further challenges in the prevention and control of antibiotic resistance posed by new CTX-M-101-like variants in Salmonella.


Introduction
The increased resistance to antibiotics in bacteria has become a global clinical and public health concern. It has been estimated that approximately 10 million people will die annually by 2050 due to drug-resistant bacteria unless there is a global response to the problem of antimicrobial resistance (AMR) [1]. Currently, cephalosporins are a common and effective first-line drug for infections by pathogens [2,3]. However, the cephalosporins resistance in Enterobacteriaceae has been frequently reported due to the abuse of this drug in clinical treatment and animal breeding [4,5]. The results from a previous study indicated that the extended-spectrum cephalosporin resistance increased from 5.46% to 12.97% between 2009 and 2016 in invasive Escherichia coli infections in hospitalized patients in the US [6]. Similar reports from China also showed that there was an increase in cefotaxime resistance from 52.2% in 2005 to 62.0% in 2014 in E. coli from inpatients and outpatients [7].

Bacterial Strains
The S. Enteritidis isolate SJTUF14523 was recovered from the stool samples of an outpatient with diarrhea in Xinjiang, China, in 2016. This isolate was identified via API20E test strips (BioMerieux, Marcy-l'Étoile, France) and serotyped via commercial antiserum (Statens Serum Institute, Copenhagen, Denmark) according to the manufacturer's guidelines. Escherichia coli (DH5α and ATCC 25922), Enterococcus faecalis ATCC 29212, and Salmonella Braenderup H9812 were also used in this study for subsequent experiments including antimicrobial susceptibility testing, gene function analysis, and S1-PFGE analysis. All strains were stored at −80 • C in Luria-Bertani (LB) broth containing 50% glycerol and propagated twice overnight before use.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed on the SJTUF14523 isolate using the agar dilution method as recommended by the Clinical and Laboratory Standard Institute (CLSI; 2019). Briefly, a stock solution (5120 µg/mL) of antibiotics was prepared and diluted to different concentrations with specific solvents according to CLSI. Then, different concentrations of antibiotics were added to sterilized Mueller-Hinton (MHA) medium cooled to 45-50 • C to prepare antibiotic plates. The S. Enteritidis SJTUF14523 cells were inoculated on the LB agar overnight culture at 37 • C. The bacteria were wiped with a sterile cotton swab and diluted in sterile normal saline. In addition, the suspension of bacteria was adjusted to approximately 0.5 McFarland turbidity and inoculated onto MHA plates without antibiotics (as a control) and with different concentrations of antibiotics for cultivation for 18-24 h at 37 • C. Antibiotic susceptibility was interpreted by using MIC values based on CLSI. The following antibiotics were tested: cefoxitin (FOX), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), meropenem (MEM), nalidixic acid (NAL), trimethoprim-sulfamethoxazole (SXT), amikacin (AMK), ampicillin (AMP), gentamicin (GEN), ciprofloxacin (CIP), kanamycin (KAN), azithromycin (AZM), ofloxacin (OFX), tetracycline (TET), chloramphenicol (CHL), and streptomycin (STR). In addition, polymyxin B (PB) and polymyxin E (PE) testing were performed with broth microdilution as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; 2019). All antibiotics used in this study were purchased from Sigma-Aldrich Shanghai Trading Co. Ltd., China. E. coli ATCC 25,922 and E. faecalis ATCC 29,212 were used as quality control isolates.

Whole-Genome Sequencing, Assembling, and Annotation
The S. Enteritidis SJTUF14523 cells were transferred to LB broth and incubated overnight at 37 • C and 200 rpm on a shaking incubator with a rotational radius of 26 mm. Genomic DNA was extracted from overnight cultures using the QIAamp DNA mini kit (Qiagen, CA). WGS was performed by the Personal Biotechnology Company (Shanghai, China) using a PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA) and the Illumina MiSeq (Illumina, San Diego, CA, USA). For the PacBio RS II platform, a 10 kbp DNA library was constructed and sequenced using single-molecule real-time (SMRT) sequencing technology. The sequence data of the PacBio RS II platform were assembled using Canu software (https://github.com/marbl/canu (accessed on 8 December 2022)) [38]. For the Illumina MiSeq platform, a 400 bp DNA library was constructed and sequenced in paired-end sequencing mode. The data from the Illumina MiSeq platform were assembled using SPAdes [39]. Finally, the consensus genome sequence was determined using Pilon software (https://github.com/broadinstitute/pilon (accessed on 8 December 2022)) [40].

Phylogenetic Analysis
The genome sequence in this study and genome sequences screened from Salmonella with cephalosporins resistance in the PATRIC database (https://patricbrc.org/ (accessed on 15 December 2022)) were used to establish phylogenetic trees. Single-nucleotide polymorphisms (SNPs) were extracted using Snippy (https://github.com/tseemann/snippy (accessed on 15 December 2022)) to generate the core genomic alignment. Gubbins was then used to identify and remove recombination regions using an algorithm that iteratively identified loci containing elevated densities of base substitutions, and then the resulting pairwise SNP differences were calculated [45]. The core SNP alignment was used to generate a maximum-likelihood phylogeny using RAxML v8.1.23 [46] with the GTR nucleotide substitution model. We also conducted 100 random bootstrap replicates to assess the node support. The display, annotation, and management of phylogenetic trees were performed using the ITOL tool [47].

Plasmid Conjugation and Transformation Experiment
Conjugation experiments were performed as previously described with E. coli C600 as the recipient [32]. Briefly, Salmonella used as the donor was incubated with the recipient overnight, mixed, and transferred to the filter membrane on an LB plate for overnight culture. Transconjugants were selected on MacConkey agar plates supplemented with cefotaxime (4 µg/mL) and rifampin (200 µg/mL). Transconjugants were further identified using 16S RNA and PCR.
For the transformation experiments, plasmid DNA from the XDR isolates was extracted using the Qiagen Plasmid Midi Kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). The purified plasmid was transformed into E. coli DH5α (Takara Biotechnology, Dalian, China) cells. The transformants were selected using MacConkey agar with cefotaxime (4 µg/mL). The MICs of the transconjugants and transformants were tested using the agar dilution method as recommended by CLSI 2019.

S1-PFGE Experiment
Pulsed-field gel electrophoresis (PFGE) with S1 nuclease (Takara Biotechnology, Dalian, China) digestion was carried out to determine the size of the plasmid. Briefly, after cells (OD600 = 0.95 − 1.00) were fixed with SeaKem Gold agarose (Cambrex BioScience, Walkersville, MD, USA) and subsequently lysed, the embedded DNAs were digested using 18 U S1 enzymes (Takara Biotechnology, Dalian, China) in a 37 • C water bath for 15 min. The restricted DNA fragments were separated in 0.5 × TAE buffer at 14 • C for 19 h using a CHEF Mapper electrophoresis system (Bio-Rad, Richmond, CA, USA) with pulse times of 2.16-63.8 s. The PFGE image was obtained with a Gel Imager System (Bio-Rad, USA). S. Braenderup H9812 was used as the DNA size marker.

Emergence of bla CTX-M-101 in S. Enteritidis Isolate
In the investigation of the ESBL CTX-M subtype in Salmonella in China, we identified that S. Enteritidis SJTUF14523 carried bla CTX-M-101 , a new CTX-M subtype. Antimicrobial susceptibility testing showed that the SJTUF14523 isolate exhibited resistance to ceftazidime (MIC = 64 µg/mL), cefotaxime (MIC = 256 µg/mL), and cefepime (MIC = 16 µg/mL) (Table 1), and this isolate was also resistant to ampicillin (MIC ≥ 128 µg/mL), nalidixic acid (MIC ≥ 128 µg/mL), trimethoprim-sulfamethoxazole (MIC ≥ 16/304 µg/mL), and kanamycin (MIC ≥ 128 µg/mL). Therefore, SJTUF14523 was an MDR isolate. Wholegenome sequencing was then performed on this isolate. Antibiotic resistance genes and chromosome point mutation were consistent with the presented resistance to β-lactams (bla CTX-M-101 and bla TEM-1b ), aminoglycosides (aac(6 )-Iaa, aph(3")-Ib, and aph(6)-Id), sulfonamides (sul2), and quinolones (gyrAD87Y) ( Table S1). Extended-spectrum cephalosporins are the primary drugs of choice for treating salmonellosis; however, there is a rising emergence of Salmonella resistance to these antibiotics due to their abuse and overuse in humans and livestock [48][49][50][51]. In 2013, the total amount of antibiotics used in China was about 162,000 tons, approximately 160 times that of the United Kingdom, and 48% of which was used for human consumption; the rest was shared by animals [52]. Furthermore, the production yields of fluoroquinolones (including ciprofloxacin) and β-lactams (including ceftriaxone) in China were estimated to be 27,300 and 34,100 tons, respectively [52]. Therefore, governmental regulations limiting the use of antimicrobial agents have been issued in China to reduce the potential threat of MDR bacteria to public health.
In this study, S. Enteritidis SJTUF14523 exhibited resistance to ceftazidime, cefotaxime, cefepime, ampicillin, nalidixic acid, trimethoprim-sulfamethoxazole, and kanamycin, indicating its MDR. Moreover, this isolate carried some antibiotic resistance genes that included the novel CTX-M type gene bla CTX-M-101 . Exploring the function of this gene is important for understanding the transmission mechanism of MDR Salmonella.

bla CTX-M-101 Mediated the Resistance to Cephalosporin
The effect of bla CTX-M-101 on cephalosporin resistance was further explored through conjugation, transformation, and gene cloning. Plasmid p14523A was successfully transferred into E. coli C600 as the recipient through conjugation. There Cephalosporin resistance is usually due to the ESBLs produced by Enterobacteriaceae [50]. The bla CTX-M gene is generally located on transferable plasmids that could facilitate the dissemination among E.coli, Salmonella, and other pathogens [50,[53][54][55]. The bla CTX-M-14 subtype has been found in Salmonella Indiana isolates from chickens and pigs in Guangdong [56] and also in Salmonella Typhimurium isolates from humans [50]. The bla CTX-M-65 subtype was the predominant type in Salmonella Indiana isolates from humans and food-producing animals in Henan [48]. To the best of our knowledge, this is the first report that bla CTX-M-101 was found in S. Enteritidis isolates, which sounds an alarm regarding the control of the emergence of new antimicrobial resistance gene variants in bacteria.

Characterization of a Novel Hybrid Plasmid Carrying blaCTX-M-101
The characterization of plasmids was further analyzed in SJTUF14523. There were three plasmids in SJTUF14523: p14523A, p14523B, and p14523C (Table S1). Furthermore, blaCTX-M-101 was located on plasmid p14523A, which possessed an IncI1-Iα plasmid structure with GC content of 50.0% and was 85,862 bp in length. The Blastn results showed that p14523A was similar to Escherichia coli pMS6192C (89% coverage; 98.87% identity), p2 (91% coverage; 98.93% identity), Salmonella Anatum PDM04 (86% coverage; 97.82% identity), and Salmonella Heidelberg p20760-1 (87% coverage; 97.81% identity). All of these plasmids harbored the IncI1-Iα replication gene and possessed similar conjugation, maintenance, and stability function regions (Figure 2a). However, variable regions containing parM and umuD genes and an insert sequence (ISEcp1) as well as an antibiotic resistance gene (blaCTX-M-101) were special to p14523A. It was interesting that these variable regions showed a high similarity to those of the chromosome sequence from S. Enteritidis SE81 but differed by blaCTX-M-55 in SE81 (Figure 2a). The SE81 chromosome sequence showed a high similarity to a plasmid structure containing conjugation, maintenance, and stability function regions, suggesting that it might have originated from plasmids. In addition, the Salmonella Infantis isolates shown in Figure 1 belonged to ST32, and this clone carried the cephalosporin resistance genes of bla CMY-2 and bla CMY-65 . Salmonella Dublin belonged to ST10, and this clone carried bla CMY-2 . Salmonella Newport mainly belonged to ST45, and this clone mainly carried bla CMY-2 . Salmonella Heidelberg belonged to ST15, and this clone mainly carried bla CMY-2 . Salmonella Typhimurium was clustered with Typhimurium var.5- (Figure 1), both of which mainly belonged to ST19, and this clone mainly carried bla CMY-2 . A part of Salmonella Typhimurium belonged to ST2076, and some belonged to ST213, both of which were uncommon STs. Salmonella Kentucky isolates were divided into two clades: one clade belonged to ST198, and another clade belonged to ST152. A variety of ESBL genes including bla CTX-M-15 , bla CTX-M-1 , bla CMY-4 , bla CMY-2 , bla CMY-16 , bla OXA-48 , and bla VIM-48 were identified in the ST198 clone. The ST152 clone mainly carried bla CMY-2 .

Characterization of a Novel Hybrid Plasmid Carrying bla CTX-M-101
The characterization of plasmids was further analyzed in SJTUF14523. There were three plasmids in SJTUF14523: p14523A, p14523B, and p14523C (Table S1). Furthermore, bla CTX-M-101 was located on plasmid p14523A, which possessed an IncI1-Iα plasmid structure with GC content of 50.0% and was 85,862 bp in length. The Blastn results showed that p14523A was similar to Escherichia coli pMS6192C (89% coverage; 98.87% identity), p2 (91% coverage; 98.93% identity), Salmonella Anatum PDM04 (86% coverage; 97.82% identity), and Salmonella Heidelberg p20760-1 (87% coverage; 97.81% identity). All of these plasmids harbored the IncI1-Iα replication gene and possessed similar conjugation, maintenance, and stability function regions (Figure 2a). However, variable regions containing parM and umuD genes and an insert sequence (ISEcp1) as well as an antibiotic resistance gene (bla CTX-M-101 ) were special to p14523A. It was interesting that these variable regions showed a high similarity to those of the chromosome sequence from S. Enteritidis SE81 but differed by bla CTX-M-55 in SE81 (Figure 2a). The SE81 chromosome sequence showed a high similarity to a plasmid structure containing conjugation, maintenance, and stability function regions, suggesting that it might have originated from plasmids.  Further analysis showed that p14523A was a hybrid plasmid through a chimera process of the p2 and SE81 chromosomes. The p2 and SE81 sequences might have formed a hybrid plasmid through interaction between a homologous region (HR), namely a 1394 bp DNA sequence encoding a hypothetical protein (Figure 2b). We proposed that two daughter plasmids (p2 and pSE81-like plasmid) were aligned at the HR sequence, then homologous recombination activities occurred and finally formed a hybrid plasmid (Figure 2c). Currently, plasmid fusion often occurs during bacterial conjugation, and this is often mediated by insertion sequences (IS26) [57,58]. However, p14523A was not formed in the conjugation, and it pre-existed in the original isolate. Therefore, the hybrid plasmid in this study was different from those in the previous studies, and it is urgent to control and prevent the dissemination of p14523A-like plasmids among Enterobacteriaceae. . IRL (TTTCCGTCAGG) and IRR (CCTGACGGAAA) were found at the end of this transposon unit, providing evidence for traces of transposon. We then proposed that the process of co-integrating into a plasmid was much more likely mediated by the transposon ISEcp1. ISEcp1 appeared to be able to use IRL in combination with a sequence beyond its IRR end to move an adjacent region, yielding the transfer of the entire transposon unit [59,60]. ISEcp1-mediated transposition of bla CTX-M-64 , bla CTX-M-2 , bla CTX-M-3 , and bla TEM-1b have been demonstrated [61][62][63]. Moreover, ISEcp1 could capture DNA regions with different sizes and simultaneously transfer adjacent regions [59]. Therefore, ISEcp1-mediated transposition could be responsible for capturing bla CTX-M-101 by the IncI1-Iα plasmid.

Conclusions
In summary, our study highlighted the emergence of blaCTX-M-101, a new blaCTX-M vari- pKP18069-CTX. Areas shaded in green indicate homologies between the corresponding genetic loci on each plasmid. Boxes or arrows represent the ORFs. Red, antibiotic resistance genes; yellow, IS/transposase; gray, hypothetical protein; blue, replicon; brown, other genes.

Conclusions
In summary, our study highlighted the emergence of bla CTX-M-101 , a new bla CTX-M variant, in S. Enteritidis. The bla CTX-M-101 gene mediated the resistance to third-generation cephalosporin (ceftazidime and cefotaxime). The bla CTX-M-101 gene was located on an IncI1-Iα transferable plasmid p14523A that facilitated its spread among Enterobacteriaceae through bacterial conjugation. This IncI1-Iα plasmid appeared to be very active and could fuse DNA fragments from other plasmids or chromosomes by activating homologous recombination. We also identified the transposition event driven by ISEcp1 in this plasmid, which was likely to be responsible for the capture and transfer of bla CTX-M-101 among different plasmids in Enterobacteriaceae. The possibility of dissemination of these CTX-M-101-like variants and their transferable plasmids among Enterobacteriaceae should be an important consideration in the "One Health" system.

Data Availability Statement:
The data of this study are available from the authors upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.