Coexistence of a nonresistance-conferring IncI1 plasmid favors persistence of the blaCTX-M-bearing IncFII plasmid in Escherichia coli

ABSTRACT The interaction between coexisting plasmids can affect plasmid-carried resistance gene persistence and spread. However, whether the persistence of the blaCTX-M gene in clinical Enterobacteriaceae is related to the interaction of coresident nonresistance-conferring plasmids has not been reported. This study was initiated to elucidate how a nonresistance-conferring IncI1 plasmid affected the blaCTX-M-bearing IncFII plasmid colocated on the same cell. Herein, we constructed three isogenic derivatives of E. coli C600, designated as C600FII, C600I1, and C600FII+I1, which harbored the blaCTX-M-IncFII plasmid and/or the nonresistance-IncI1 one. We discovered that strain C600FII+I1 conferred higher fitness advantages than strain C600FII; also, the stability of the blaCTX-M-IncFII plasmid was noticeably improved in an antibiotic-free environment when it coexisted with the IncI1 plasmid. To further explore why the IncI1 plasmid enhanced the persistence of the blaCTX-M-IncFII plasmid, we assessed the blaCTX-M-IncFII plasmid's copy numbers, conjugation frequencies, and rep gene expressions in strains C600FII and C600FII+I1. The results demonstrated that the rep expressions of the blaCTX-M-IncFII plasmid in strain C600FII+I1 was greatly decreased, along with the plasmid’s copy numbers and mating efficiencies, compared to those in strain C600FII. Moreover, further study revealed that the intracellular ATP levels of strain C600FII+I1 were far lower than those of strain C600FII. Our findings confirmed that coexistence of the nonresistance-IncI1 plasmid can keep the blaCTX-M-IncFII plasmid more stable by increasing the fitness advantages of the host bacteria, which will pose a threat to preventing the long-term presence of the plasmid-carried blaCTX-M gene in clinical Enterobacteriaceae. IMPORTANCE So far, plasmid-carried blaCTX-M is still the most common extended-spectrum beta-lactamase (ESBL) genotype in clinical settings worldwide. Except for the widespread use of third-generation cephalosporins, the interaction between coexisting plasmids can also affect the long-term stable existence of the blaCTX-M gene; however, the study on that is still sparse. In the present study, we assess the interaction of coinhabitant plasmids blaCTX-M-IncFII and nonresistance-IncI1. Our results confirmed that the increased fitness advantages of strain C600FII+I1 were attributable to the cohabitant nonresistance-IncI1 plasmid, which largely reduced the intracellular ATP levels of host bacteria, thus decreasing the rep gene expression of the blaCTX-M-IncFII plasmid, its copy numbers, and mating efficiencies, while the higher fitness advantages of strain C600FII+I1 enhanced the persistence of the blaCTX-M-IncFII plasmid. The results indicate that the nonresistance-IncI1 plasmid contributes to the long-term existence of the blaCTX-M-IncFII plasmid, implying a potentially new strategy for controlling the spread of resistance plasmids in clinical settings by targeting nonresistance plasmids.

T he production of extended-spectrum beta-lactamases (ESBLs) is the main factor leading to the resistance of Gram-negative bacteria to β-lactam antibiotics (1).Until now, bla CTX-M has emerged as the most prevalent ESBL genotype in clinical Enterobac teriaceae worldwide (2).Conjugative plasmids, specifically IncFII-type plasmids, have a crucial role in the development and horizontal dissemination of bla CTX-M (3).IncFII plasmids are widely distributed in Enterobacteriaceae, such as in E. coli, (4) and they help the host bacteria adapt to various environments, conditions, and pressures (5,6).Although resistance plasmids confer potential benefits to the host, they also impose burdens (fitness costs), manifesting as reduced growth rates and competitiveness of strains carrying plasmids under conditions where plasmid-encoded resistance genes are not selected (7), while the increased cost of plasmid adaptation and the potential loss of plasmids during bacterial cell division severely reduce plasmid persistence (7).In addition, plasmid-host genome interactions also affect the evolution of plasmid persistence (8).
Recent studies suggested that a sufficiently high transfer rate could appropriately compensate for the adaptive cost and plasmid loss (9,10).Therefore, suppressing conjugation for disrupting the persistence of resistance plasmids is an attractive and effective strategy.Many factors, such as bacterial genetic backgrounds, plasmid copy numbers, and interactions with other resistance plasmids, could affect the conjugation of antibiotic resistance plasmids (11,12).For example, Haudiquet et al. reported that conjugation rates depend on the capsule serotype of both donor and recipient strains.(13).Meanwhile, increasing evidence suggests that the interactions of multiple resistance plasmids could change the persistence of plasmid-harbored resistance genes (14,15).Gama et al. found that interactions between plasmids could determine the mode of antibiotic resistance dissemination in bacterial populations (16,17), while Dionisio and his team demonstrated that the transfer rate of plasmids was affected by the presence of different plasmids in recipient cells (18).However, it has not been reported whether the persistence of the bla CTX-M gene in clinical E. coli is related to the interaction of coinhabitant nonresistance plasmids.
In the survey of antimicrobial-resistant bacterial isolates in 2021, E. coli LWY24 was reported from feces of healthy chicken in Henan Province, China (19).The isolate LWY24 harbored three conjugative plasmids, pLWY24J-3 (bla CTX-M-55 -bearing IncFIItype, MN702385), pLWY24Jmcr-1 (mcr-1-carrying IncI2-type, MN689940), and pLWY24J-4 (nonresistance-conferring IncI1-type), which could transfer horizontally through plasmid conjugation independently, in pairs or three together.In 2022, we investigated the interaction between the bla CTX-M -bearing IncFII plasmid and the mcr-1-carrying IncI2 plasmid coexisting in the same strain (20).The findings confirmed that coresident IncFII-type and IncI2-type plasmids in E. coli were stably persistent, conferred more fitness advantages, and were easier to transfer and cotransfer.In addition, it was discovered that the mating efficiency of the bla CTX-M -IncFII plasmid decreased by 3.467 × 10 4 fold when coexisting with the nonresistance-IncI1 plasmid.However, the correla tion between the two plasmids remains unclear.This study was initiated to explore the underlying mechanism that the nonresistance-conferring IncI1 plasmid affected the bla CTX-M -bearing IncFII plasmid colocated on the same bacterial host.

The plasmids had no effect on bacterial growth
To explore the effects of plasmids FII and I1 on bacterial growth, we plotted the growth curves of strains E. coli C600, C600 FII , C600 I1 , and C600 FII+I1 based on the optical density at 600 nm (OD 600 ) values after 14-h assessment (Fig. 1).The results showed that there were no significant differences in growth among bacteria, indicating that the plasmids FII and/or I1 did not impair the growth of the bacterial host.

Plasmid I1 improved the stability of the coexisting plasmid FII
When multiple plasmids are present in the same bacterium, interactions between them can affect their transmission and persistence (15,21).In order to plot the stability-time curve of the acquired plasmids, we propagated the isogenic strains, C600 FII , C600 I1, and C600 FII+I1 culture, in antibiotic-free medium for 2 weeks (Fig. 2).The results showed that the single plasmid FII was moderately stable, remaining at around 84% on day 14, which coincided with our previous findings (20).However, when plasmids FII and I1 cohabited in the recipient cell, the stability of plasmid FII was obviously improved, with a total loss of about 8.0% (P = 0.0047) at the end, implying that plasmid I1 could improve the persistence of the coexisting plasmid FII.

Coharboring plasmids FII and I1 endow cells with fitness advantages
To determine the relative carriage costs of plasmids FII and I1, competition assays in vitro were carried out among strains C600 FII , C600 FII+I1, and C600 I1 (Fig. 3).The outcome revealed that there was no significant difference in the fitness costs between strains C600 FII and C600 I1 as well as between strains C600 FII+I1 and C600 I1 , indicating that plasmid FII would not endow any additional fitness costs with its host cell when transfer ring to strain C600 I1 .On the contrary, the isogenic strain C600 FII+I1 significantly outcom peted C600 FII from day 4 (RF = 1.155 ± .035,P = 0.0148) to day 6 (RF = 1.489 ± .011,P = 0.0001), indicating that plasmid I1 could increase the fitness advantages of host bacteria when entering strain C600 FII .Thus, E. coli co-carrying plasmids FII and I1 present higher fitness advantages than bacteria carrying single plasmid FII, which helps the bla CTX-M -IncFII plasmid maintain long-term persistence.

The reduced copies of plasmid FII were due to the coexistence of plasmid I1
To verify whether copy numbers of plasmid FII in strain C600 FII+I1 decreased, we conducted qPCR according to our previous research (20).The findings exhibited that the copy numbers of plasmid FII in strain C600 FII+I1 were 0.467 ± 0.09 per cell (Table 1), which were extremely reduced compared to those in strain C600 FII (1.773 ± 0.16 per cell) (P < 0.001), implying that plasmid I1 inhibited coexisting plasmid FII copies.Generally, the entry of plasmids into new bacterial hosts via conjugative transmission can increase fitness costs (22).Therefore, for long-term survival in the environment, plasmids have evolved corresponding adaptive compensation mechanisms to compensate for FIG 1 Growth curves of E. coli C600 and its isogenic derivatives, C600 FII , C600 I1 , and C600 FII+I1 .The strains C600 FII , C600 I1 , and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the plasmids bla CTX-M -IncFII and/or nonresistance-IncI1.
Curves indicated the mean of results from three independent experiments, and error bars denoted standard deviations for each timepoint.
the increased costs caused by plasmid entry and expression (7).Previous reports have proven that reducing plasmid copies is the best way to directly reduce the costs of survival ( 9).In the present study, the copy numbers of plasmid FII were dramatically reduced under the action of the coexisting plasmid I1, thereby increasing the fitness advantages of bacterial hosts and enhancing the persistence of the bla CTX-M -IncFII plasmid.

Plasmid I1 depressed coresident plasmid FII transfer
The conjugation rates of plasmid FII were measured using C600 FII and C600 FII+I1 as a donor and E. coli J53 as a recipient (Table 2).The mating rate of plasmid FII in the donor C600 FII was (9.650 ± 3.437) ×10 −2 , which was consistent with the findings in our previous study (20).Moreover, in the donor C600 FII+I1 , two types of transconjugants can be isolated on MacConkey agar containing cefotaxime, one harboring only a single plasmid FII at (1.869 ± 0.294)×10 −6 per donor cell and the other co-carrying plasmids FII and I1 with frequencies of (9.195 ± 4.065)×10 −7 .In total, the conjugation rates of plasmid FII in the donor C600 FII+I1 , including independent transfer and co-transfer with plasmid I1, were (2.783 ± 0.288) ×10 −6 per donor cell, which were approximately 3.467 × 10 4 -fold lower than those in the donor C600 FII .Thus, the horizontal transfer of plasmid FII can be severely inhibited by plasmid I1 coexisting in the same bacterial host.
Usually, high mating efficiencies contribute to plasmid persistence, but increase plasmid fitness costs (22).However, in our study, the mating efficiency of plasmid FII in strain C600 FII+I1 sharply decreased, compared to that of strain C600 FII , although it presented more stable.Gama et al. reported that those affecting fitness favor plasmid persistence more than those affecting conjugative transfer (14).Stevenson et al. also proved that in environments that experience occasional pulses of positive selection, even low rates of conjugation can enhance plasmid survival by enabling low-level persistence during the periods between pulses (23).Thus, we speculate that the lower plasmid conjugation rates of strain C600 FII+I1 contribute to the fitness advantages of bacterial hosts, thereby improving the persistence of plasmid FII.

The invasion of plasmid I1 enhances a long-time persistence of strain C600 FII+I1
Plasmid persistence could comprehensively be evaluated by comparing the abilities of single-plasmid and co-located plasmids in invading plasmid-free populations (24).As shown in Fig. 4A, plasmid FII could invade rapidly and appeared in most cells after 24 h, and its numbers not only remained unchanged over time but were significantly higher than those of plasmid-free bacteria.Considering that the individual plasmid FII was slightly unstable, we speculated that the higher mating efficiency of plasmid FII could help offset the loss of plasmid FII.Interestingly, when E. coli C600 and C600 FII+I1 were coincubated, plasmids FII and I1 could successfully invade bacteria either independently or together, thereby obtaining three types of plasmid-harbored bacteria, C600 FII , C600 I1, and C600 FII+I1 (Fig. 4B).Specifically, when cultured for 24 hours, strain C600 FII was 2.91 × 10 8 CFU/mL, C600 FII+I1 was 2.05 × 10 6 CFU/mL, while strain C600 I1 was the least at that time, only 3.34 × 10 5 CFU/mL, indicating the lowest invasion rates of plasmid I1.It was worth noting that strain C600 FII decreased extremely during the incubation period of 24 h to 48 h; on the contrary, strain C600 FII+I1 was found to apparently increase, implying that plasmid I1 could continue to invade strain C600 FII .Further study showed  a All data were obtained from at least three biological replicates and presented as mean ± SD.Different letters marked with the shoulder mark in the same column indicate significant differences (a vs b, P < 0.001).The strains C600 FII , C600 I1 , and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the plasmids bla CTX-M -IncFII and/or nonresistance-IncI1.
that C600 FII+I1 exhibited stability in the mixed populations after 48 hours of culture, which was consistent with the aforementioned results of plasmid stability assays.Given its lower plasmid conjugation rates, we speculated that the improved persistence of strain C600 FII+I1 was mainly attributable to the invasion of plasmid I1, which enhanced its fitness advantages.

Coexistence of plasmids FII and I1 did not change the swimming motility of the bacterial host
It is generally believed that close contact is required between the donor and recipi ent before plasmid conjugative transmission; therefore, changing the bacterial motility characteristics can affect the transfer frequency of plasmids (25).The swimming motility assay was fulfilled in the isogenic strains C600 FII and C600 FII+I1 (Fig. 5).Obviously, both strains C600 FII and C600 FII+I1 could form an initial-inoculation-point-centered approxi mate circle on the swimming plates, and there was no available difference in their swimming motility.From this, we speculated that the inhibition of coexisting plasmid I1 on plasmid FII transfer was not achieved by affecting the motility of the bacterial host.

Plasmid I1 decreased the rep gene expressions of co-resident plasmid FII
To determine whether the reduced conjugation rate of plasmid FII in strain C600 FII+I1 was mediated by changes in rep gene expression levels, we examined the mRNA expression levels of repA, repA1, repA2, and repA4 encoded by the plasmid FII (MN702385) (19).a Experiments were performed three times, and results are shown as mean ± SD.Different letters marked with the shoulder mark in the same column indicate significant differences (a vs b, P < 0.001).The strains C600 FII and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the single bla CTX-M -IncFII plasmid or coinhabitant plasmids bla CTX-M -IncFII and nonresistance-IncI1.
Except for repA2, the levels of repA, repA1, and repA4 in strain C600 FII+I1 were remarkably decreased (P ≤ 0.0036), compared to those of strain C600 FII (Fig. 6).Rep protein is a plasmid-encoded replication initiator, which is essential and rate-limiting for plasmid replication initiation and conjugative mobilization (26).Therefore, the inhibition of coexisting plasmid I1 on plasmid FII transfer is closely related to its repression of rep gene expressions in plasmid FII.

The intracellular ATP levels decreased in strain C600 FII+I1
Conjugative plasmids in E. coli are transferred from one bacterium to another through a membrane-associated macromolecular channel called the type IV secretion system (T4SS) (21).ATP plays an important role in conjugation by providing energy for T4SS assembly and substrate transport, plasmid replication, and transport (27,28).So we further measured the intracellular ATP levels in strains C600 FII+I1 and C600 FII .
As expected, the fluorescence intensities in strain C600 FII+I1 were markedly reduced, compared to strain C600 FII (P < 0.0001) (Fig. 7), indicating that the intracellular ATP levels of strain C600 FII+I1 sharply decreased.Thus, we hypothesized that the decrease of intracellular ATP levels may be one of the reasons for the decrease of rep gene expression and mating efficiency of plasmid FII in strain C600 FII+I1 .

Conclusion
Taken together, the higher fitness advantages of strain C600 FII+I1 enhance its harboring bla CTX-M -IncFII plasmid persistence.While more fitness advantages of strain C600 FII+I1 are attributable to the cohabitant nonresistance-conferring IncI1 plasmid that largely reduces the copies and mating efficiencies of the bla CTX-M -IncFII plasmid by inhibiting its rep gene expression, which was repressed by decreasing the intracellular ATP levels of host bacteria.Our findings demonstrate that the advantages are evident in the coexistence of plasmids bla CTX-M -IncFII and nonresistance-conferring IncI1 in E. coli, which can advance the long-term clinical presence of the bla CTX-M resistance gene and pose a threat to controlling its spread.
In the past, the interaction between plasmids mainly focused on the coexistence of resistant plasmids (16,18) and almost ignored whether nonresistance plasmids would have an effect on the co-inhabitant resistance one.In the present study, we found that the nonresistance IncI1 plasmid could improve the cohabitant bla CTX-M -IncFII plasmid fitness advantages and persistence by reducing its copies and mating efficiencies.As a result, it may be a potential new strategy that influencing resistance plasmid persistence in clinical settings by targeting certain nonresistance plasmids in the future.

FIG 5
The swimming motility of strains C600 FII and C600 FII+I1 .Experiments were performed three times, and the results are shown as mean ± SD. "a" indicates that there is no significant difference in swimming mobility between strains C600 FII+I1 and C600 FII .The strains C600 FII and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the single bla CTX-M -IncFII plasmid or coinhabitant plasmids bla CTX-M -IncFII and nonresistance-IncI1.

Growth kinetics and plasmid stabilities
Growth curve assays were performed according to the method described in our previous study (20).The E. coli C600 FII ,C600 I1, and C600 FII+I1 cultures grew in a tube at 37°C with shaking and diluted 1:100 into fresh media every 24 h for 336 h to investigate the stability of their plasmids (29).Viable counts were performed at 0, 48, 96, 144, 192, 240, 288, and 336 h.For every timepoint, dilutions of the cultures were plated to nonselective and cefotaxime-containing media.Cells not containing the FII plasmid were killed by cefotaxime-containing media, and cells containing I1 plasmids were verified by PCR using the prime nusG (a NusG family protein in plasmid I1).The stability of plasmids was calculated by the following formula : plasmids-carrying cells counts / antibiotic-free plate counts.

In vitro fitness assays
A competition experiment was conducted with the strains C600 FII , C600 I1 , and C600 FII+I1 in fresh LB broth, following the method described in previous studies (29).Appropriately diluted samples of competition mixtures were plated onto LB agar containing 4 mg/L of cefotaxime and antibiotic-free at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h to determine total bacterial count.Further, each colony was amplified by PCR using nusG primers to verify whether it carried plasmid I1.Finally, the relative fitness (RF) was calculated as previous (20).Experiments were repeated in at least three separate assays.

Quantitative real-time PCR (qPCR)
The copy numbers of plasmids in strains C600 FII , C600 I1 , and C600 FII+I1 were measured by qPCR operate according to the instructions of ChamQ Universal SYBR qPCR Master Mix (Nanjing Vazyme Biotech Co., Ltd.).The genes bla CTX-M , nusG, and dxs (1-deoxy-D-xylose-5-phosphate synthase, as a housekeeping gene) were cloned into pUC19 to generate pUC19-bla CTX-M , pUC19-nusG, and pUC19-dxs.Thereafter, they were used as template DNA and bla CTX-M -q, nusG-q, and dxs-q as primers (Table 3) for qPCR amplifications, and corresponding standard curves were established.Soon afterward, the genomic DNA of strains C600 FII , C600 I1, and C600 FII+I1 was extracted using the HiPure Bacterial DNA Kit (Takara, China), and qPCR was performed with the aforementioned primers.The copy numbers of plasmids per cell were calculated as bla CTX-M /dxs and nusG/dxs ratios.The experiment was performed in triplicate.

Mating assays
The donor strains C600 FII and C600 FII+I1 were re-transferred to recipient E. coli J53 AZ R .Briefly, standing overnight cultures were diluted 1:200 in fresh LB broth and incubated with shaking at 37°C to an OD 600 of 0.5.Thereafter, the donor and the recipient were mixed in pairs in a 5.0 mL total volume at a ratio of 1:1 and mated for 4 h at 37°C.Mating was stopped by cooling the samples on ice for 1 min and vortexing vigorously for 1 min.Then, each sample was serially diluted in 0.9% NaCl and plated onto LB agar supplemen ted with 4 mg/L cefotaxime.The plates were subsequently incubated overnight at 37°C FIG 7 The intracellular ATP levels of the isogenic derivatives C600 FII and C600 FII+I1 .The strains C600 FII and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the single bla CTX-M -IncFII plasmid or coinhabitant plasmids bla CTX-M -IncFII and nonresistance-IncI1.Compared to C600 FII , strain C600 FII+I1 had significantly lower ATP levels (P < 0.001).All data were obtained from at least three biological replicates and presented as mean ± SD.Significance levels are indicated by the numbers of asterisks (****P < 0.001).and counted.Colonies of C600 FII+I1 and E. coli J53 mixture were amplified to verify whether it co-harbored plasmids FII and I1.Each experiment was performed at least three times.Thereafter, mating efficiency was calculated and evaluated.

Plasmid invasion assays
The plasmid invasion assay was conducted following the previous study by the Yang team with some modifications (30).Briefly, overnight cultures of E. coli C600 were diluted 100-fold into 2 mL of fresh antibiotic-free LB broth and mixed with the 100,000-fold dilutions of C600 FII or/and C600 FII+I1 at a ratio of 1:1.The LB broth was replaced every 24 hours, and an equal portion of the samples was plated onto LB agar containing rifampin (400 µg/mL) or rifampin (400 µg/mL) +cefotaxime (4 µg/mL) to determine the viable counts after gradient dilutions.The plates were then incubated under appropriate conditions, and the viable bacterial counts were determined through gradient dilutions.All viable bacteria were able to grow on the plates contained rifampin, and bacteria containing bla CTX-M -IncFII were also able to grow on the media supplemented with rifampin +cefotaxime.Further, PCR was used to distinguish C600 FII , C600 I1, and C600 FII+I1 by the specific gene bla CTX-M and nusG.All invasion assays were replicated three times biologically.

Swimming motility assay
The single colonies C600 FII , C600 I1, and C600 FII+I1 were cultured in fresh LB broth overnight at 37°C at 180 RPM and then transferred to fresh LB broth, cultured to an OD 600 of 0.5.Bacteria were point-inoculated in the swimming assay plate containing 0.3% agar with a sterile toothpick.Then, the plates were incubated at 37°C for 28 h.The motility ability was assessed by examining the movement distance of the bacteria away from the inoculation point (16).The experiment was performed with three biological replicates.

Real-time relative quantitative PCR (RT-qPCR)
Based on whole-genome sequencing and comparative analysis, plasmid FII encodes the genes repA, repA1, repA2, and repA4.Therefore, the expression levels of all rep genes in strains C600 FII and C600 FII+I1 were assayed using RT-qPCR (19).Primer sequences for RT-qPCR are listed in Table 3.A single colony of each strain was cultured in the LB medium at 37°C.After overnight growth, the cultures were diluted 1:100 in fresh LB broth and grown to an OD 600 of 0.5.Thereafter, the total bacterial RNA was extracted using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Bio, Inc., Shiga, Japan).The quantity and purity of the extracted RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Hvidovre, Denmark) by measuring the A 260 absorption and calculating the A 260 /A 280 ratio.The cDNA samples were synthesized from the extracted RNA using a cDNA reverse transcription kit with gDNA Eraser (TaKaRa Bio, Inc.).RT-qPCR was then performed according to our previous study (20).The relative expression levels in C600 FII+I1 compared to C600 FII were calculated using the 2 −ΔΔCT method.Three independent biological replicates were conducted for each experiment.

Intracellular adenosine triphosphate (ATP) levels
Intracellular ATP levels of strains C600 FII , C600 I1 , and C600 FII+I1 were assessed using an Enhanced ATP Assay Kit (Beyotime, Shanghai, China).Overnight cultures of strains were removed and resuspended to obtain an OD 600 of 0.5.Bacterial pellets were lysed by lysozymes, and the luminescence of the supernatant was determined using the Spark 10 M Microplate reader (Tecan).The experiment was performed at least in triplicate.

Statistical analyses
The statistical analyses were performed with GraphPad Prism v8.0.2 (GraphPad Software).Data were presented as mean ± standard deviation (SD) of three replications.Mean values between groups were compared using unpaired t-test, and P < 0.05 was considered to be statistically significant.

FIG 2
FIG 2The stability of plasmids IncFII and IncI1 in strains C600 FII , C600 I1 , and C600 FII+I1 .The strains C600 FII , C600 I1 , and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the plasmids bla CTX-M -IncFII and/or nonresistance-IncI1.Data shown are the means of results from three independent assays, and error bars represented the standard deviation of the mean (n = 3).

FIG 3
FIG 3Pairwise competition assays between strains C600 FII , C600 I1 , and C600 FII+I1 .The strains C600 FII , C600 I1 , and C600 FII+I1 were isogenic derivatives of E. coli C600, which harbored the plasmids bla CTX-M -IncFII and/or nonresistance-IncI1.Each boxplot represents the distribution of relative fitness values for each timepoint: the horizontal line in the boxplot is the median, and the bottom and top of the boxplot show the lowest and the highest values, respectively.Asterisks denote significant differences using unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.005).

FIG 4
FIG 4 Plasmid invasion assays of plasmids bla CTX-M -IncFII and IncI1.(A) Cocultures with E. coli C600 and C600 FII .(B) Cocultures with E. coli C600 and C600 FII+I1 .Calculate viable bacteria of co-cultures with plasmid-free and plasmid-containing E. coli C600.Plasmid-free E. coli were mixed with a 10 −3 -fold excess of plasmids containing single plasmid bla CTX-M -IncFII and colocated plasmids bla CTX-M -IncFII and IncI1 at the beginning of the invasion assay.Every dot indicated the average value of three biological replicates.E. coli C600 is represented by hollow circles, C600 FII is represented by hollow triangles, C600 I1 is represented by hollow diamonds, and C600 FII+I1 is represented by hollow squares.

Li
Yuan http://orcid.org/0000-0001-5602-6910Natural Science Foundation of China (NSFC) 32373074 Li Yuan Innovative research Team in the university of Henan province 23IRTSTHN021 Li Yuan Key projects of the Natural Science Foundation of Henan province 232300421111 Li Yuan

TABLE 2
The transfer rates of the IncFII-type plasmid in strains C600 FII and C600 FII+I1

TABLE 3
The primers used in the study