Dissemination of Ceftriaxone-Resistant Salmonella Enteritidis Harboring Plasmids Encoding blaCTX-M-55 or blaCTX-M-14 Gene in China

Salmonella Enteritidis was the primary foodborne pathogen responsible for acute gastroenteritis. The growing ceftriaxone resistance poses a significant threat to public health. Infection with S. Enteritidis has emerged as a major public health concern, particularly in developing countries. However, research on ceftriaxone-resistant S. Enteritidis (CRO-RSE) remains limited, particularly concerning its resistance mechanism, plasmid structure, and transmission characteristics. This study aims to address these gaps comprehensively. We collected 235 S. Enteritidis isolates from Hangzhou First People’s Hospital between 2010 and 2020. Among these, 8.51% (20/235) exhibited resistance to ceftriaxone. Whole-genome analysis revealed that 20 CRO-RSE isolates harbored blaCTX-M-55 or blaCTX-M-14 on the plasmid. Moreover, the dissemination of the blaCTX-M-type gene was associated with IS26 and ISEcp1. Plasmid fusion entailing the integration of the p1 plasmid with antibiotic resistance genes and the p2 (pSEV) virulence plasmid was observed in certain CRO-RSE. Additionally, the structural analysis of the plasmids unveiled two types carrying the blaCTX-M-type gene: type A with multiple replicons and type B with IncI1 (Alpha) replicon. Type B plasmids exhibited superior adaptability and stability compared to type A plasmids within Enterobacteriaceae. Interestingly, although the type B (S808-p1) plasmid displayed the potential to spread to Acinetobacter baumannii, it failed to maintain stability in this species.


Introduction
Salmonella Enteritidis can contaminate eggs leading to asymptomatic infections in birds [1], which subsequently spread to humans, posing a significant threat to global health [2].Salmonella primarily infects humans via the intestinal route and can manifest in a variety of diseases, with acute gastroenteritis being the most prevalent.This can result in either mild or fulminant diarrhea [3], often accompanied by low-grade fever, nausea, and vomiting.Presently, the Salmonella genus comprises two species: S. bongori and S. enterica.S. enterica is further categorized into six subspecies: enterica I, salamae II, arizone IIIa, diarizone IIIb, houtenae IV, and indica VI [4].S. enterica I is predominantly responsible for human infections [5].According to serotyping, S. enterica I can be further subdivided into S. Typhimurium, S. Paratyphi, S. Enteritidis, and so on.Among these, S. Enteritidis is a notable non-typhoid Salmonella strain capable of causing foodborne illnesses with a significant global impact on human health that cannot be overlooked [6].
Ceftriaxone, a vital antibiotic, is utilized in the treatment of S. Enteritidis infections.Resistance to ceftriaxone typically arises from the production of extended-spectrum β-lactamases (ESBLs), which possess the ability to hydrolyze expanded-spectrum cephalosporins, such as cefotaxime, ceftriaxone, ceftazidime, and cefepime, as well as monobactams like aztreonam [7].Historically, the predominant families of ESBLs were the TEM and SHV enzymes.However, since the early 2000s, CTX-M enzymes have emerged as the most commonly encountered ESBLs [8].CTX-M enzymes are classified as class A ES-BLs and encompass multiple variants that currently dominate ESBL prevalence in clinical and community settings [9].According to the annual surveillance data from the China Antimicrobial Surveillance Network (http://www.chinets.com/Data/AntibioticDrugFast,accessed on 14 March 2023 and 14 March 2024), 380 and 503 strains of S. Enteritidis exhibited resistance to ceftriaxone, accounting for 12.4% and 20.7% of resistance in 2022 and 2023, respectively.These data suggest a notable increase in ceftriaxone resistance within S. Enteritidis, a development that urgently calls for attention.
Plasmids serve as crucial vectors for the transmission of ESBLs, especially conjugable plasmids, which play a pivotal role in ESBL dissemination.Plasmids harboring ESBLs often contain numerous mobile genetic elements, such as ISEcp1, ISCr1, and IS26, which are associated with the transfer and transmission of ESBLs [10].For instance, IS26 facilitates the dissemination of antibiotic resistance genes (ARGs) through the conservative reaction and by forming translocatable units (TUs) [11].Moreover, IS26 can be inserted upstream of a resistance gene, creating hybrid promoters that enhance the expression of resistance genes.Previous studies have identified that IS26 was involved in forming the promoter responsible for bla BES-1 expression as observed through sequence analysis of the IncP6-type plasmid (12,957 bp) in Serratia marcescens [12].
As a significant foodborne pathogen, S. Enteritidis contributes to ceftriaxone resistance.The emergence of ceftriaxone-resistant strains not only poses a threat to global public health and safety but also presents new therapeutic challenges.Therefore, understanding the related resistance mechanisms and transmission characteristics is paramount for public health.In this study, we elucidated the ceftriaxone resistance mechanism, plasmid characteristics, and plasmid transmission traits of ceftriaxone-resistant S. Enteritidis (CRO-RSE) isolated from Hangzhou First People's Hospital, Zhejiang Province, from 2010 to 2020.We employed scientific research methods, including plasmid gene analysis, antibiotic sensitivity testing, conjugation experiments, growth curve analysis, and plasmid stability assessment.These findings provide a crucial research foundation for the surveillance and control of CRO-RSE.

Analysis of Plasmid Structure Harboring bla CTX-M-14
The S808 AGRs were located on plasmids.S661 carrying bla CTX-M-14 matched to S808-p1 (Figure S3a).S114-p1 carrying bla CTX-M-55 exhibited a structure similar to that of S808-p1, which carries bla CTX-M-14 .Furthermore, S808-p1 shared a similar structure with plasmids from the National Center for Biotechnology Information (NCBI), including the unnamed plasmid in Salmonella enterica from ground turkey in the USA (CP022064, coverage 85%, identity 99.30%), the plasmid p5848A2 identified in E. coli from the Netherlands (LR743516, coverage 85%, identity 99.31%), and the plasmid pESBL20160056 detected in E. coli found in Homo sapiens in Denmark (MH472638, coverage 85%, identity 99.30%) (Figure S3b).This suggests that both bla CTX-M-55 and bla CTX-M-14 are carried as passenger genes within IncI1-type plasmids during their propagation.

Plasmid Type Classification in This Study
For subsequent plasmid studies, the plasmids detected by CRO-RSE were categorized into two types: type A and type B (93 kbp).Type A comprised subtypes A1 (130-170 kb) and A2 (93 kb).Subtype A1 plasmids, represented by S750-p1 and S161-p1, harbored bla CTX-M-55 and virulence genes, along with four replicons: IncX1, IncFII (pHN7A8), IncFIB (S), and IncFII (S).Subtype A1 plasmids contained four parts (parts a, b, c, and d) of the T4SS genes (Figure 2a).The differences between parts c and d included the absence of traV in part d and the truncation of traJ in part c.Additionally, S750-p1 lacked parts a and b of the T4SS compared to S161-p1.Furthermore, the analysis of plasmid fusion events revealed that the S204-p1 plasmid was derived from a portion of the S161-p1 plasmid.The subtype A2 plasmids, including S204-p1, S131-p1, S273-p1, S379-p1, and S664-p1, carried bla CTX-M-55 and two replicons, IncX1 and IncFII (pHN7A8).Subtype A2 plasmids contained three parts (parts a, b, and d) of the T4SS genes (Figure 2a).Additionally, S664-p1 lacked the b and d parts of the T4SS genes compared to the other subtype A2 plasmids.Type B plasmids, represented by S808-p1 and S114-p1, carried bla CTX-M-55 or bla CTX-M-14 and one replicon, IncI1 (Alpha).Type B plasmids contained part e of the T4SS genes (Figure S3b), which differed from parts a, b, c, and d.Both type A and B plasmids harbored the origins of transfer (oriT), relaxase, coupling protein (T4CP), and T4SS.Plasmids containing these four parts were considered conjugable plasmids.

Results of Plasmid Conjugation
The results of plasmid conjugation are shown in Table 2. Susceptibility testing of transconjugants revealed a significant increase in recipient resistance to ceftriaxone upon conjugation of plasmids containing the bla CTX-M-type gene (Table 3).Subtype A2 plasmids were capable of conjugating with RrifSL1344, RrifJ53, and RrifATCC13883.Despite carrying oriT, relaxase, T4CP, and T4SS, S131-p1 failed to conjugate to any of the four recipients in our experiment.Furthermore, compared to other subtype A2 plasmids, S664-p1 could only conjugate to E. coli J53, potentially limiting its conjugative capability owing to the absence of a portion of the T4SS (absence of b and d).Similarly, compared to other subtype A1 plasmids, S750-p1 could only conjugate to S. typhi SL1344, likely because of the absence of a portion of the T4SS (absence of a and b).Additionally, S161 carrying a subtype A1 plasmid, S379 carrying a subtype A2 plasmid, and S808 carrying a type B plasmid were selected as donor bacteria, and RrifJ53 served as the recipient bacterium to calculate the conjugation frequency.The results revealed that the conjugation efficiencies of S161-p1, S379-p1, and S808-p1 were 1.65 × 10 −6 , 0.75 × 10 −4 , and 9.09 × 10 −4 , respectively.In summary, none of the subtype A1 plasmids were able to conjugate to K. pneumoniae ATCC13883, unlike the subtype A2 plasmids.Moreover, the conjugation efficiency of the subtype A2 plasmid was 45 times higher than that of the subtype A1 plasmid.Consequently, subtype A2 plasmids exhibited greater transmission potential than subtype A1 plasmids, implying that the absence of virulence genes and ARGs promoted plasmid dissemination.Regarding type B plasmids, both S114-p1 and S808-p1 were capable of conjugating with S. typhi SL1344, E. coli J53, and K. pneumoniae ATCC13883.Notably, the type B plasmid (S808-p1) could conjugate with Acinetobacter baumannii ATCC17978, indicating its potential to spread to A. baumannii.Furthermore, the conjugation efficiency of the type B plasmid was 12 times higher than that of the subtype A2 plasmid, indicating that the type B plasmid (S808-p1) had greater transmission capacity than the type A plasmid.The first column in parentheses represents the MIC of ceftriaxone for the recipient, while the ceftriaxone MICs of the donors were all higher than 256 µg/mL.The rows correspond to the recipients, with the columns indicating the conjugated plasmids, and the numbers denote the MIC of ceftriaxone for the transconjugants (µg/mL).The recipients included rifampicin-induced S. Typhimurium SL1344 (RrifSL1344), E. coli J53 (RrifJ53), K. pneumoniae ATCC13883 (RrifATCC13883), and A. baumannii ATCC17978 (RrifATCC17978).

Fitness Cost Analysis of Different Plasmids under Different Strain Backgrounds
Subtype A1 plasmids exhibited an increased fitness cost in various Enterobacteriaceae species (SL1344 and J53) (Figure 3a).Subtype A2 plasmids displayed varying fitness costs across different genera of Enterobacteriaceae (SL1344, J53, ATCC13883) (Figure 3b).Overall, within Enterobacteriaceae, certain transconjugants carrying the subtype A2 plasmids exhibited no fitness cost, whereas all those harboring the subtype A1 plasmids displayed an increased fitness cost.This suggests that the subtype A2 plasmids have better adaptability than the subtype A1 plasmid within Enterobacteriaceae.Additionally, type B plasmids increased the fitness costs only in E. coli J53 (Figure 3c).Consequently, the type B plasmids exhibit better adaptability than the type A plasmids.

Discussion and Conclusions
In this study, we investigated 20 strains of CRO-RSE collected from Hangzhou First People's Hospital, Zhejiang Province.Our findings revealed a close association between the spread of blaCTX-M-type genes and the presence of IS26 and ISEcp1 elements.Analysis of the plasmid structure indicated a highly conserved fundamental backbone, with regions carrying ARGs showing frequent alterations.These ARG-containing regions consistently harbored one or several mobile elements (eg.IS26 and ISEcp1), suggesting multiple insertion or recombination events due to mobile elements.Consequently, these regions could act as hotspots for homologous recombination [13].A study reporting on S. enterica demonstrated that IS26-mediated composite transposon may facilitate the dissemination of blaCTX-M-55 [14].Moreover, numerous studies on different serotypes of S. enterica also

Discussion and Conclusions
In this study, we investigated 20 strains of CRO-RSE collected from Hangzhou First People's Hospital, Zhejiang Province.Our findings revealed a close association between the spread of bla CTX-M-type genes and the presence of IS26 and ISEcp1 elements.Analysis of the plasmid structure indicated a highly conserved fundamental backbone, with regions carrying ARGs showing frequent alterations.These ARG-containing regions consistently harbored one or several mobile elements (eg.IS26 and ISEcp1), suggesting multiple insertion or recombination events due to mobile elements.Consequently, these regions could act as hotspots for homologous recombination [13].A study reporting on S. enterica demonstrated that IS26-mediated composite transposon may facilitate the dissemination of bla CTX-M-55 [14].Moreover, numerous studies on different serotypes of S. enterica also observed the transmission of bla CTX-M-type genes in association with IS26 and ISEcp1 [15][16][17][18].Additionally, ceftriaxone-resistant isolates S161 and S750, collected in 2013 and 2020, respectively, exhibited a unique plasmid configuration where two plasmids harboring virulence genes and ARGs were fused, mediated by IS26.The emergence of such fusion plasmids, encoding both ARGs and virulence genes, presents a novel challenge to public health.Therefore, continuous monitoring of IS26-mediated transmission of significant pathogens is essential.In 2021, a study reported the presence of two hybrid plasmids mediated by IS26 and Tn6952 in S. Enteritidis [19].However, this study did not assess the conjugative ability of plasmids through conjugation experiments.
A previous study in Guangdong, China, reported that the plasmids IncI1 and IncFII were likely the primary replicons contributing to the prevalence of bla CTX-M-55 in E. coli [20].Additionally, previous reports indicated a close association between IncF-type plasmids and ESBL-producing strains of E. coli [21].In our study, replicons IncI1(Alpha), IncFII(S), In-cFII(pHN7A8), IncFIB(S), and IncX1 were associated with the spread of bla CTX-M-55.Several previous studies reported that the IncI1 plasmids carrying the bla CTX-M-type genes conferred resistance to ceftriaxone in Salmonella [3,15,22].In this study, we observed a similar trend.Importantly, we found, for the first time, that the IncI1 plasmid carrying the bla CTX-M-type gene had the potential to spread across Enterobacteriaceae to A. baumannii through conjugation experiments.However, its stability in A. baumannii was limited, persisting for only approximately 5 days.While conjugation and growth curve experiments indicated that the Inc1 (Alpha)-type plasmid (type B plasmid) exhibited superior characteristics over type A plasmids in terms of transmissibility and fitness cost, it was noteworthy that the presence of type B plasmids (only three strains) was not prevalent among the 20 CRO-RSE samples collected from 235 S. Enteritidis.However, given the advantages of Inc1 (Alpha)-type plasmids in terms of transmission capacity and fitness cost, they are likely to emerge as primary vectors for the transmission of the bla CTX-M-type gene in the future, warranting further investigation into their activities.

Collection of Isolates and Clinical Information
A total of 235 strains of S. Enteritidis were isolated from patients across various clinical departments of Hangzhou First People's Hospital between 2010 and 2020.Colonies with correct characteristics, such as colorless transparency, with or without a black center, were selected from SS agar and streaked to ensure purity.Subsequently, the selected strains were amplified and resuspended in 20% glycerol broth before being stored in a −80 • C freezer for subsequent experiments.Serological agglutination tests were conducted using the Salmonella Diagnostic Serum Test Kit (Ningbo Tianrun Biopharmaceutical Company Limited, Ningbo, China) for the identification of S. Enteritidis.Clinical data, including collection date, gender, age, place of origin, department, and disease, were retrieved from the hospital's network system, with identifiers concealed to ensure privacy.

Bacterial Isolate Identification and Antibiotic Susceptibility Test
All 235 isolates were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry systems (Bruker Daltonics, Bremen, Germany).Antibiotic susceptibility testing was conducted for all collected isolates, covering azithromycin, ampicillin, cefoxitin, imipenem, cotrimoxazole, carbenicillin, tetracycline, ceftriaxone, and ciprofloxacin, through the disk diffusion method.Additionally, the MICs of transconjugants and 20 CRO-RSE, which include ceftriaxone, were determined using the broth dilution method.Interpretation of the results was based on the 2023 Clinical and Laboratory Standards Institute guidelines (CLSI) [23] and the 2022 European Committee on Antimicrobial Susceptibility Testing [24].

Plasmid Transfer Assays
Nine strains (S204, S131, S273, S379, S664, S161, S750, S114, and S808) served as donor bacteria, which underwent short-read and long-read sequencing.Recipients comprised RrifSL1344, RrifJ53, RrifATCC13883, and RrifATCC17978.Donor plasmids carrying the bla CTX-M-type gene were transferred to the recipients.Donors and recipients were cultivated in 2 mL of Mueller Hinton Broth (MHB) for four hours at 37 • C on a shaker.Subsequently, 100 µL of both donors and the recipients were mixed, and 20 µL of the mixture was spotted onto a 0.22 µm membrane (Merck, Darmstadt, Germany) affixed to Mueller Hinton Agar (MHA), followed by overnight incubation at 37 • C. The next day, the bacteria on the membrane were dissolved in MHB, and 20 µL was spotted onto a double antibiotic plate, which was then incubated overnight at 37 • C. Transconjugants were selected on a double antibiotic plate containing rifampicin (1000 mg/L) and ceftriaxone (64 mg/L).Antibiotic sensitivity testing results were interpreted according to CLSI guidelines (2023) [11].The presence of bla CTX-M-type genes, replicon genes, and Salmonella's housekeeping gene aroC in transconjugants was confirmed via PCR.Transconjugants were also identified using MALDI-TOF.Conjugation frequency was calculated as the number of transconjugants per donor.

Bacterial Growth Curve Assay
Three single colonies of both recipients and transconjugants were selected and inoculated into MHB, with blank controls set up simultaneously.Ten microliters of bacterial solution were added to 1 mL of MHB culture medium, equivalent to a 1:100 dilution of the overnight bacterial culture.This diluted solution was then added into a honeycomb plate, with 200 µL per well, conducting three technical replicates for each sample while simultaneously establishing a blank control.The honeycomb plate was then placed in an automatic growth curve instrument (Growth Curves, Turku, Finland).The parameters were set to 37 • C, and the optical density at 600 nm (OD600) was measured every 5 min for a total of 20 h.The growth rate during the logarithmic phase was calculated, and the relative growth rate of the strain was determined using R script [38], with the recipients serving as the reference.

Plasmid Stability Assay
After overnight culture, the bacterial solution was diluted 1:1000 with 2 mL of MHB medium and incubated overnight with shaking at 200 rpm.This process was repeated daily up to the 20th generation.During this time, bacterial solutions from the 5th, 10th, 15th, and 20th generations were diluted onto MHA plates, and 48 single colonies were streaked onto both an antibiotic plate containing ceftriaxone (64 mg/L) and an MHA plate to verify ceftriaxone-resistant plasmids.Transconjugants that grew on both ceftriaxone plates and MHA were considered positive for plasmid retention, while those that could not grow on ceftriaxone plates but grew on MHA were considered negative for plasmid retention.The plasmid loss rate was calculated using the following formula: Plasmid loss rate = (mean number of negative clones from three replicates/48) × 100.

Figure 1 .
Figure 1.Molecular characteristics of CRO-RSE.(a) Core-genome phylogeny of 20 CTX-M-type-positive S. Enteritidis strains, based on the reference sequence of S. Enteritidis S114.The tree scale indicates the number of allelic differences.(b) Comparative analysis of ten CRO-RSE isolates, identified solely via short-read sequencing, carrying the bla CTX-M-55 gene, using S204-p1 as the reference sequence.

Figure 2 .
Figure 2. Plasmid fusion or cleavage event of two plasmids.(a) Fusion event observed at S204, S131, S273, S379, and S664 with reference to S161.The details of a-d parts are explanated in Table 2. (b) Detailed illustration of the fusion mechanism using S204 as an example.(c) Plots depicting the fusion patterns of the two plasmids.(d) Plots illustrating the pattern of intramolecular replicative transposition in S161-p1.

Figure 2 .
Figure 2. Plasmid fusion or cleavage event of two plasmids.(a) Fusion event observed at S204, S131, S273, S379, and S664 with reference to S161.The details of a-d parts are explanated in Table 2. (b) Detailed illustration of the fusion mechanism using S204 as an example.(c) Plots depicting the fusion patterns of the two plasmids.(d) Plots illustrating the pattern of intramolecular replicative transposition in S161-p1.

Figure 3 .
Figure 3.Comparison of relative growth rates of recipients carrying subtype A1 (a), A2 (b), and Btype (c) plasmids.(d) Plasmid stability experiments.The x-axis represents the number of generations of transconjugants, while the y-axis represents the number of transconjugants that still contain S808-p1.ns : p value ≥ 0.05, not significant; * : p value is from 0.01 to 0.05, significant; ** : p value is from 0.001 to 0.01, very significant; *** : p value < 0.001, very significant.

Figure 3 .
Figure 3.Comparison of relative growth rates of recipients carrying subtype A1 (a), A2 (b), and B-type (c) plasmids.(d) Plasmid stability experiments.The x-axis represents the number of generations of transconjugants, while the y-axis represents the number of transconjugants that still contain S808-p1.ns: p value ≥ 0.05, not significant; *: p value is from 0.01 to 0.05, significant; **: p value is from 0.001 to 0.01, very significant; ***: p value < 0.001, very significant.

Table 3 .
Results of ceftriaxone susceptibility testing in the transconjugants.