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Article

Intra- and Interspecies Conjugal Transfer of Plasmids in Gram-Negative Bacteria

by
Julia R. Savelieva
,
Daria A. Kondratieva
and
Maria V. Golikova
*
Department of Pharmacokinetics & Pharmacodynamics, Gause Institute of New Antibiotics, 11 Bolshaya Pirogovskaya Street, 119021 Moscow, Russia
*
Author to whom correspondence should be addressed.
Biomedicines 2025, 13(1), 238; https://doi.org/10.3390/biomedicines13010238
Submission received: 29 December 2024 / Revised: 15 January 2025 / Accepted: 17 January 2025 / Published: 20 January 2025
(This article belongs to the Special Issue Infectious Diseases: Prevention, Diagnosis, and Treatment Under Antimicrobial Resistance Global Urgency)
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Abstract

:
Background/Objectives: Plasmid-mediated resistance is a significant mechanism that contributes to the gradual decrease in the efficacy of antibiotics from various classes, including carbapenems. The aim of this study is to investigate the frequency of transfer of carbapenemase-encoding plasmids from K. pneumoniae to E. coli and P. aeruginosa. Methods: Matings were performed on agar with subsequent isolation of transconjugant, recipient, and donor colonies. The frequency of conjugation (CF) and minimum inhibitory concentrations (MICs) of meropenem were determined for the PCR-confirmed transconjugants. A pharmacodynamic study was conducted using a hollow-fiber infection model on E. coli transconjugant in order to evaluate its viability in the presence of therapeutic concentrations of meropenem. Results: CF for K. pneumoniae-K. pneumoniae was similar to that for K. pneumoniae-E. coli and was higher the higher was meropenem MIC of the K. pneumoniae donor. The meropenem MICs for K. pneumoniae and E. coli transconjugants were higher (0.25–4 μg/mL) compared to recipients (0.03–0.06 μg/mL). P. aeruginosa did not acquire plasmids from K. pneumoniae. In pharmacodynamic experiments, an E. coli transconjugant with MIC of 2 mg/L within the “susceptibility range”, failed to respond to meropenem treatment. Conclusions: The frequency of conjugation between K. pneumoniae and E. coli falls within a similar range. A higher permissiveness of K. pneumoniae for plasmids from K. pneumoniae, i.e., within the same species, was observed. Conjugation did not occur between K. pneumoniae and P. aeruginosa. The transconjugants with meropenem MICs with borderline susceptibility may pose a potential threat to the efficacy of meropenem.

1. Introduction

Horizontal gene transfer, in particular via plasmids through conjugation, is a major mechanism that contributes to the spread of antibiotic-resistant bacteria around the world [1,2]. However, after nearly a century of research, there is still much to be learned about the conjugation of bacteria. It is crucial to enhance our understanding of this process, as the spread of plasmids carrying antibiotic resistance genes among different species of Gram-negative bacteria is thought to be a major cause of infections that pose a threat to global health. Previous studies have demonstrated that the efficacy of conjugation is influenced by a variety of factors, including the individual characteristics of the donor and recipient cells, the bacterial species involved [3], the levels of antimicrobial resistance, the number of plasmids present in the donor cell [4], and the conjugative permissiveness of the recipient [5].
Carbapenems, which are among the most commonly prescribed antimicrobial agents for a wide range of clinical indications, are at risk of becoming ineffective due to the emergence of resistant strains carrying plasmids with carbapenemase genes [6]. Among the bacterial species commonly found to carry carbapenemase genes encoding carbapenem resistance, Klebsiella pneumoniae is one of the most prevalent. Various diseases, including bloodstream infections, pneumonia, and urinary tract infections, can be caused by K. pneumoniae [7]. Individuals with weakened immune systems are particularly vulnerable to serious infections caused by this bacterium. Treating infections caused by these strains has become more challenging due to their increased resistance to antibiotics. The most widespread carbapenemases, including KPC, OXA-48 and NDM, are associated with this species [8,9]. The range of bacteria that can exist in the natural environment of K. pneumoniae and participate in conjugation processes is quite wide; one of the most likely partners for plasmid transfer is Escherichia coli. K. pneumoniae inhabits the same environment as E. coli and their interaction is common. Pseudomonas aeruginosa often occurs alongside K. pneumoniae and E. coli at the same location. In addition, P. aeruginosa is known to cause serious hospital-acquired infections that can be difficult to treat, particularly due to producing carbapenemases. Several studies have reported the isolation of clinical strains of P. aeruginosa carrying KPC carbapenemase genes [10,11].
The focus of our study was to investigate the efficiency of conjugation between various bacterial species, both closely and distantly related. We investigated K. pneumoniae, E. coli, and P. aeruginosa, as well as conducting a pharmacodynamic evaluation of meropenem against a transconjugant strain generated through mating experiments. The aim was to assess its viability in the presence of an antibiotic, simulating its clinical dosing regimen using a hollow-fiber infection model (HFIM). The HFIM is an effective tool for studying antimicrobial efficacy and has numerous applications, including investigating bacterial resistance and assessing the clinical efficacy of antimicrobials [12]. In this study, epithelial lining fluid (ELF) meropenem pharmacokinetics [13] was simulated following the administration of 2 g every 8 h in a 3 h infusion during 5-day treatments in an HFIM.
The investigation of issues related to the conjugation efficiency between clinically relevant strains of Gram-negative bacteria, assessing their relative potential to acquire plasmids with carbapenemase genes, and subsequently replicating them in order to survive under meropenem exposure, determines the clinical significance of this study.

2. Materials and Methods

2.1. Antimicrobial Agents, Bacterial Strains and Susceptibility Testing

Meropenem and CCCP (3-chlorophenylphenhydrazone) powders were purchased from Sigma-Aldrich (St. Louis, MO, USA). Three clinical isolates of K. pneumoniae were used as plasmid donors in mating experiments: 38, 485, and 565 (Table 1).
Meropenem-susceptible carbapenemase-non-producing strains of E. coli K-12 C600, E. coli ATCC 25922, K. pneumoniae ATCC 700603, K. pneumoniae 188 (clinical isolate), P. aeruginosa ATCC 9027, and P. aeruginosa ATCC 27853 were used as recipients in mating experiments. Plasmid donors [14,15] and the recipient strains K. pneumoniae 188 and E. coli C600 were kindly provided by Dr. Ageevets A.V., Pediatric Research and Clinical Center for Infectious Diseases, Saint Petersburg, Russia.
Susceptibility testing was carried out using broth microdilution techniques with a standard inoculum of approximately 5 × 105 CFU/mL. Meropenem MICs were determined according to standard recommendations using cation-supplemented Mueller–Hinton broth (CSMHB) (Becton Dickinson, Franklin Lakes, NJ, USA) [16]. Before reading, microplates were incubated at 37 °C for 18–20 h. MIC values in each case were obtained at least in triplicate, and modal MICs were estimated.
The susceptibility of P. aeruginosa isolates to meropenem was also tested in the presence of the efflux pump inhibitor CCCP (3-chlorophenylphenhydrazone) at a concentration of 50 μg/mL using the standard susceptibility testing technique. The efflux pump inhibitor solution was freshly prepared before every experiment and every test was carried out on a 96-well plate. Additionally, it has been verified that the growth of all P. aeruginosa strains is not inhibited in the presence of 50 µg/mL of an inhibitor. Resistance due to the efflux pumps was presumed if the MIC of the meropenem decreased by 4-fold or more in the presence of the inhibitor [17].

2.2. Mating Experiments

The protocol of mating experiments followed to obtain transconjugants is summarized in Figure 1 [18].
To distinguish between donor, recipient and transconjugant cells, all the recipient strains (E. coli C600, E. coli ATCC 25922, K. pneumoniae ATCC 700603, K. pneumoniae 188, P. aeruginosa ATCC 9027, and P. aeruginosa ATCC 27853) were incubated on the media (three passages) with rifampicin (at concentrations from 50 to 250 µg/mL) to produce rifampicin-resistant mutants. These mutants have MICs of rifampicin greater than 512 µg/mL. For all experiments, these rifampicin-resistant mutants were used.
Bacteria were grown in Luria broth (LB, Becton Dickinson, Franklin Lakes, NJ, USA) and Luria agar (LA, Becton Dickinson, Franklin Lakes, NJ, USA) media at 37 °C. When required, LA was supplemented with antimicrobial agents at the following final concentrations (µg/mL): meropenem (0.5–2) and rifampicin (150–200).
Matings were performed overnight on the LA plates according to a previously published protocol with minor modifications [18]. Briefly, the 1:1 mixture of the donor and recipient in the late logarithmic growth phase was plated on the LA surface and incubated at 37 °C for 18–20 h. The mixed growth was then scraped from the plate surface and resuspended in 1 mL of saline, and to quantify the numbers of donor, recipient, and transconjugant cells, the cell mixture was diluted as appropriate. Subsequently, 100 µL samples were spread on appropriate selective plates with meropenem and/or rifampicin at the following final concentrations: meropenem (equal to 4×MIC of recipient) and rifampicin (150–200 µg/mL). Parent strains were plated in parallel with the matings and then processed similarly to the matings as controls. Isolated colonies from matings presumed as transconjugant and parental strains from controls were used to identify recombinants and parental forms. The conjugation efficiency was assessed by the conjugation frequency (CF): ratio of the number of CFU of transconjugants per mL to the number of CFU of recipients plus transconjugants per mL [19]. The data corresponding to the absence of conjugation as confirmed by the PCR are depicted as equivalent to a limit of detection for conjugation frequency of 10⁻⁹.
“Possible” transconjugants were screened by streaking colonies from the selection plate with meropenem and rifampicin onto a fresh plate with the same antibiotics to look for growth. The plasmid acquisition by recipient strains was confirmed by the PCR with primers specific to genes encoding plasmid replication proteins and relaxases (Table S1). In addition, a PCR was performed to confirm or rule out the presence of carbapenemase genes in potential transconjugant strains. A PCR was performed according to a standard protocol for amplification of fragments with a size of 1 kb [20].

2.3. In Vitro Dynamic Model and Operational Procedure Used in the Pharmacodynamic Experiments

The HFIM was used to evaluate meropenem pharmacodynamics and to conduct growth control experiments. The flowchart for the pharmacodynamic experiments is presented in Figure 2.
The studies were performed using a hollow-fiber bioreactor (Fresenius dialyzer, model AV400S, Fresenius Medical Care AG, Bad Homburg, Germany) that represents the infection site (HFIM schematic illustration is provided in Figure S1). The operational procedure is described in detail elsewhere [21]. Briefly, antibiotic dosing and sampling were processed automatically, using computer-assisted controls. The system was filled with sterile CSMHB and placed in an incubator at 37 °C. The inoculum of an 18 h culture of E. coli was injected into the hollow-fiber bioreactor to produce a bacterial concentration of 108 CFU/mL. After a 2 h incubation, samples were obtained to determine the starting bacterial concentration; then, the infusion of CSMHB with antibiotic was initiated. The duration of each experiment was 120 h. To verify the reliability of pharmacokinetic simulations, throughout each experiment the bioreactor was sampled immediately after the end of infusion (3 h) and at the 6th hour of the dosing interval.
In each experiment, the bacteria-containing medium from the central unit of the model was sampled to determine bacterial concentrations throughout the observation period. Samples (100 µL) were serially diluted as appropriate and 100 µL was plated onto Mueller–Hinton agar plates, which were placed in an incubator at 37 °C for 24 h. The lower limit of accurate detection was 1 × 102 CFU/mL (equivalent to 10 colonies per plate).
To monitor the time courses of antibiotic-resistant subpopulations of K. pneumoniae in the pharmacodynamic experiments, the central unit of the model was multiply sampled throughout the observation period (120 h). The samples were serially diluted, if necessary, plated on Mueller–Hinton agar (MHA) with 2×, 4×, 8× and 16×MIC of meropenem, and incubated for 24–48 h at 37 °C. The viable counts were screened visually for growth. The lower limit of detection was 10 CFU/mL (equivalent to at least one colony per plate).

2.4. Antibiotic Dosing Regimens and Simulated Pharmacokinetic Profiles

Meropenem treatment mimicked the therapeutic dosing regimen: 2000 g administered every 8 h, as a 3 h intravenous infusion. A mono-exponential profile in epithelial lining fluid (ELF) after thrice-daily dosing of meropenem with a half-life (t1/2) of 1.4 h was simulated [13] for five consecutive days with a total of 15 infusions. The pharmacokinetic parameter values were as follows: CMAX = 32.4 µg/mL, 24-h area under the concentration–time curve (AUC) = 375 (µg × h)/mL. Before all pharmacodynamic simulations, the system was calibrated and preliminary in vitro pharmacokinetic experiments in CSMHB without bacteria were conducted.

2.5. Statistical Analysis

The reported MIC data were obtained by calculation of the respective modal values. The resultant conjugation frequency was calculated as arithmetic mean ± standard deviations for three replicate experiments. The data from each group were analyzed for statistically significant differences (p < 0.05) in the data mean values between the groups using SigmaPlot 12 statistical software (Systat Software Inc., headquartered in San Jose, CA, USA) by a paired two-sample t-test.
In pharmacodynamic and growth control experiments, bacterial count data were calculated as arithmetic mean ± standard deviations for three replicate experiments. Based on these data, kinetic growth and time-kill curves were constructed.

3. Results

3.1. Donor and Recipient Strains, Meropenem and Rifampicin Susceptibility

In mating experiments, three K. pneumoniae donor strains were used. These strains carried plasmids with carbapenemase genes (blaKPC-2 or blaOXA-48) and varied in resistance to meropenem. As recipients, meropenem-susceptible bacterial strains from different species that do not produce carbapenemases were used. These included closely related strains of E. coli and K. pneumoniae, both members of the Enterobacteriaceae family, and P. aeruginosa. Table 2 presents the MICs of meropenem for bacterial strains tested.

3.2. Mating Experiments and Meropenem Susceptibility of Transconjugants

The flowchart in Figure 3 illustrates the data on paired strains used in the mating experiments and the meropenem susceptibility of the resulting transconjugant strains.
As described in the Materials and Methods section, confirmation of the transconjugants was performed by plating different transconjugant colonies on agar plates supplemented with meropenem and rifampicin and by performing PCR analysis. E. coli transconjugant colonies growing on agar plates containing meropenem (plus rifampicin), regardless of the plasmid donor, were difficult to cultivate in the presence of meropenem due to the fitness cost. Only a few samples maintained steady growth after repeated cultivation. PCR analysis of E. coli transconjugants confirmed plasmid carriage in only 25% of the samples (Supplementary Figure S2). Only these colonies were used to calculate the conjugation frequencies in K. pneumoniae-E. coli pairs. These colonies exhibited good growth on media with or without meropenem and retained their plasmids during 15 passages. In contrast, K. pneumoniae transconjugants showed steady growth when cultivated on agar with meropenem (plus rifampicin), and plasmid carriage was confirmed in 100% of the samples (during 15 passages).
We should pay special attention to the results obtained from the mating of K. pneumoniae with P. aeruginosa. The “possible” transconjugants of P. aeruginosa seemed to grow well on agar with meropenem (plus rifampicin) and were further successfully cultivated in the presence of antibiotic. However, according to PCR analysis, the percentage of plasmid carriage among these colonies was equal to 0% (Figure S2). Given the potential for P. aeruginosa to transform carbapenemase genes from plasmids and use them to resist meropenem, we conducted PCR analysis to detect the presence of specific genes in these isolates. The primers used to detect carbapenemase genes are presented in Table S1 of the Supplementary Materials. As a result, PCR confirmed the absence of blaKPC-2 and blaOXA-48 genes in potential transconjugants, indicating that transformation had not occurred, and these strains relied on their intrinsic ability to resist meropenem, using efflux pumps, for example. To verify this assumption, we determined meropenem MICs for all P. aeruginosa isolates in the absence and presence of an efflux pump inhibitor, CCCP (3-chlorophenylphenhydrazone) (Table 3). Based on the results obtained, it was found that resistance to meropenem in three of six isolates is due to the functioning of efflux pumps. However, other P. aeruginosa isolates did not show a decrease in the meropenem MIC in the presence of CCCP.
All transconjugants from matings of K. pneumoniae with either K. pneumoniae or E. coli, which appeared to be steadily growing on agar with meropenem and were confirmed to carry plasmids, were then tested for meropenem susceptibility. As shown in Figure 3, transconjugants of K. pneumoniae and E. coli, isolated after mating with the donor K. pneumoniae 565 (carrying a plasmid with the blaKPC-2 carbapenemase genes), had higher meropenem MICs compared to those isolated after mating with donors K. pneumoniae 485 and 38 (both carrying plasmids with the blaOXA-48 carbapenemase genes). Specifically, in the first case (donor K. pneumoniae 565) for K. pneumoniae transconjugants meropenem MICs were 4 µg/mL (up to a 132-fold MIC increase compared to the recipient), while in the second case (donors K. pneumoniae 485 and 38) MICs were 0.25 and 0.5 µg/mL (up to a 16-fold MIC increase compared to the recipient). Similarly, in the first case (donor K. pneumoniae 565) for E. coli transconjugants meropenem MICs were 1–2 µg/mL (up to a 64-fold MIC increase compared to the recipient) while in the second (donors K. pneumoniae 485 and 38)-0.25 and 0.5 µg/mL.
With P. aeruginosa, unlike the isolated colonies did not carry plasmids, we evaluated the meropenem MICs for them as they could grow on the agar with meropenem. As shown in the flowchart, these isolates appeared to have meropenem MICs of 4–8 µg/mL.
In order to provide a more comprehensive characterization of isolated transconjugant strains of K. pneumoniae and E. coli, and to gain an understanding of how their susceptibility profiles had changed following conjugation, we assessed each isolate for the expression of plasmid-encoded resistance elements (Tables S2–S4). As can be seen from the Tables, resistance to antibiotics in transconjugants compared to recipients is caused by genes located on plasmids. In the case of pOXAAPSS1/2 plasmids, only resistance to beta-lactams was transferred to initially susceptible recipient strains, while in the case of pKPCAPSS, resistance to both beta-lactam and fluoroquinolones was transferred, as well as resistance to macrolides.

3.3. Conjugation Frequency

The results of matings were analyzed by determining the conjugation frequency as the ratio of number of transconjugants confirmed by PCR to the number of recipients (data summarized in Supplementary Table S5). In general, the conjugation frequency was similar between K. pneumoniae and K. pneumoniae or E. coli and varied from 10−7 to 10−3 or from 10−7 to 10−4, respectively. In order to investigate specific aspects of the plasmid transfer between donors and recipients, we generated a histogram, which is illustrated in Figure 4. We compared the frequency of conjugation between the donor strains of K. pneumoniae and the recipient groups. As can be seen from the graph, for the recipient E. coli and K. pneumoniae strains, the conjugation rates differ depending on the donor strain of K. pneumoniae. When K. pneumoniae 38 acted as the donor, the conjugation frequency (of ~10−7) was relatively lower than that for K. pneumoniae 565 (ranging from ~10−5 to ~10−4) and K. pneumoniae 485 (of ~10−6) donors. The conjugation frequency increased depending on the donor in the following order: K. pneumoniae 38 < 485 < 565. Therefore, for each recipient strain, the highest rate of plasmid transfer was observed when the donor strain was K. pneumoniae 565. The rate was then higher when the donor was K. pneumoniae 485, compared to K. pneumoniae 38, with which the lowest rate of transfer was recorded. Among P. aeruginosa, no similar trends were observed, as the conjugation could not be detected. Described differences were statistically significant (p < 0.05).

3.4. Pharmacodynamic Evaluation of E. coli Transconjugant Strain

As was previously shown, transconjugants of K. pneumoniae and E. coli with increased MICs were obtained in mating experiments. The MICs were higher for K. pneumoniae (4 μg/mL instead of 0.03–0.06 μg/mL in the recipient) and E. coli (2 μg/mL instead of 0.03 μg/mL in the recipient), which carry plasmids containing blaKPC–2 carbapenemase genes.
In the context of the emergence of carbapenem-resistant bacterial strains in clinical settings and the decreasing efficacy of carbapenems, it is crucial to investigate whether meropenem monotherapy still maintains its efficacy against these bacteria, despite their ability to produce carbapenemases. In this light, the pharmacodynamic study was designed to demonstrate how transconjugant strains are able to resist meropenem, when its concentrations correspond to those found in clinical settings and are constantly changing, as occurs in humans in the infection site. In other words, this aspect of the study allows us to anticipate and understand the chance of such strains surviving after they arise from contact with a plasmid donor (using the benefits of a new plasmid containing carbapenemase genes), and subsequently being exposed to antibiotics. For this purpose, we conducted pharmacodynamic experiments with meropenem and the KPC carbapenemase-producing E. coli transconjugant strain C600/565, with a meropenem MIC corresponding to the “susceptibility” breakpoint of 2 µg/mL (according to EUCAST guidelines) and with the E. coli C600 recipient that served as a control. In these experiments, the pharmacokinetics of meropenem observed in the ELF after a high-dose regimen of 2 g every 3 h was simulated.
The results of simulations with recipient strain E. coli C600 and transconjugant strain E. coli C600/565 are shown in Figure 5.
As seen from the figure, the numbers of E. coli C600/565 (pKPCAPSS transconjugant) increased rapidly after an initial 6 h decline; bacterial regrowth was accompanied by the intensive selection of meropenem-resistant cells. The recipient strain E. coli C600 was found to be eliminated from the bioreactor within the first 6 h. That is, initially, this strain without plasmid was unable to develop meropenem resistance.

4. Discussion

As the result of our investigation, we detected that overall conjugation frequency in pairs K. pneumoniae-K. pneumoniae, and K. pneumoniae-E. coli varied in a wide range from high (~10−3) to low (~10−7). Apparently, plasmid transfer is dependent on a combination of internal features as well as recipient and donor strains. It is known that many factors can influence these processes, such as plasmid-encoded regulatory elements, the specificity of interaction between host chromosomes and plasmids, and the external environment [23]. In addition, conjugation dynamics may depend on both the plasmid acquisition cost and the fitness cost [24], existence of a specific recipient cell surface for conjugative transfer [25], trade-off between lag times, and growth rate [26].
When analyzing the histograms presented in Figure 4, we identified that the meropenem resistance level of the donor was consistent with the effectiveness of the horizontal gene transfer in pairs K. pneumoniae-K. pneumoniae and K. pneumoniae-E. coli. One possible explanation for the higher conjugation rates in donors with greater antibiotic resistance is that they may have a larger number of plasmids per cell compared to less resistant donors. This could make them more efficient at conjugation. However, there is also evidence to suggest an inverse relationship between the number of plasmid copies per cell and the size of the plasmid [27,28]. This information suggests that, in our study, the strain K. pneumoniae 565, with the largest plasmid (127,970 bp), may have fewer copies compared to other strains (K. pneumoniae 485 and 38), with smaller plasmids (63,359 bp and 66,284 bp, respectively) [29]. Consequently, the number of plasmid copies may not fully explain why the highest conjugation rate was observed in the donor strain, which presumably has a lower number of copies, or why the rates also differed between pairs with donors, K. pneumoniae strains 485 and 38, both carrying plasmids of similar sizes. Additional factors may influence the conjugation frequency, such as the carbapenemase gene doses [30] and level of their expression [31], and the carbapenem enzyme activity [32,33]. It is likely that a combination of these factors contributes to the outcome.
It is well known that carbapenem-hydrolyzing enzymes such as KPC carbapenemases are more efficient at degrading carbapenem antibiotics compared to OXA-48 carbapenemases [32,33]. Interestingly, transconjugants carrying blaKPC-2 genes (K. pneumoniae and E. coli) demonstrated higher levels of resistance to meropenem compared to transconjugants carrying blaOXA-48 carbapenemases as measured by their MICs (2–4 µg/mL versus 0.25–0.5 µg/mL) (Figure 3). This may be due to the individual plasmid properties and their acquisition cost and/or fitness cost [24] in recipient cells. It is worth noting that we observed a fitness cost in all E. coli transconjugants. In addition, multiple carbapenemase gene copies in the transconjugant plasmids may also contribute to the higher meropenem MICs [30].
Unlike K. pneumoniae and E. coli, P. aeruginosa exhibited distinctive characteristics that set it apart. “Potential” transconjugants were not observed to acquire plasmids, so conjugation did not occur. We would like to discuss the issue of whether these colonies may be false transconjugants–small satellite colonies that may grow around the beta-lactamase-producing colonies as a halo [34]. In the case of P. aeruginosa, it is difficult to assume the presence of satellite colonies based on the absence of any carbapenemase-producing colonies and observation of distinct, well-developed large colonies that continue to grow well in medium containing meropenem (Figure S3).
Figure 3 illustrates the fact that, regardless of the donor, the MIC values for P. aeruginosa colonies that were selected in mating experiments but did not carry plasmids remain high, ranging between 4 and 8 µg/mL. This observation suggests that P. aeruginosa would transform carbapenemase genes or employ additional resistance mechanisms beyond plasmid conjugation and expressing of carbapenemase genes in the presence of meropenem. In fact, the P. aeruginosa is characterized by a flexible genome and the ability to implement various antibiotic resistance mechanisms except for carbapenemase production, such as efflux pumps and the modifications in the expression and/or structure of porins [35,36]. However, in our study carbapenemase genes in isolated colonies were not detected using PCR; efflux pump functioning was detected in three of six P. aeruginosa isolates. Therefore, in order to gain a better understanding of the complex mechanisms that lead to resistance development in P. aeruginosa during their interaction with K. pneumoniae, it is essential to expand our research to include a larger number of donor–recipient pairs. Furthermore, a more in-depth genetic analysis is necessary.
Additionally, we would like to discuss our findings regarding the ability of transconjugants to further grow in a medium containing meropenem. As it turns out, not all E. coli transconjugant colonies were capable of subsequent growth. In fact, E. coli and P. aeruginosa “possible” transconjugants did not contain plasmids immediately following the initial cultivation in 75% and 100% of cases, respectively (Figure S2). Slowly growing E. coli colonies isolated from matings were unlikely to be satellites, as they did not have a characteristic “satellite” phenotype similar to P. aeruginosa (Figure S3), appeared to be separated from each other, and had almost a normal size. However, these colonies were not taken into account when calculating conjugation frequencies, in order to avoid generating inaccurate results. Nevertheless, among these colonies, we identified variants with stable plasmids. When these plasmid-containing variants were cultivated in media with or without meropenem for over 15 passages, they exhibited good growth, retained their plasmids, and were characterized by stable meropenem MICs during periodic assessments (at least 30 times).
The exception was K. pneumoniae transconjugants that all acquired the plasmids successfully and replicated them with further cultivation. The recent study on K. pneumoniae and E. coli demonstrated that, when acquiring the same plasmid from a K. pneumoniae donor, the K. pneumoniae strain was more conjugation-permissive than the E. coli [5,37]. This also applies to our results, where K. pneumoniae recipients exhibit 100% plasmid permeability, unlike E. coli and, especially, P. aeruginosa. The phenomenon of plasmid loss or non-permissiveness, which we assume was inherent in E. coli and P. aeruginosa isolates, has been previously described and is a common occurrence among bacteria during cell division [38]. Various factors can contribute to plasmid loss in bacterial cells. Several possible explanations for this have been suggested, ranging from a metabolic burden introduced by plasmid replication and the expression of plasmid-encoded genes to disruption of essential host genes due to integration of plasmid DNA, alteration in host gene regulation, and other metabolic consequences, such as the introduction of novel efflux pumps that could potentially remove important biomolecules from the cell [39]. For instance, the host genetic background may play an important role in determining the fitness of plasmids. That is to say, a particular plasmid may decrease fitness in one strain with a specific genetic background, have no noticeable effect on fitness in another, or even be beneficial in yet another strain [38].
It is worth noting that among transconjugant strains with relatively high (2–4 µg/mL) or low (0.25–0.5 µg/mL) meropenem MICs, E. coli variants with blaKPC-2 carbapenemase genes and moderate meropenem MICs equal to 1–2 µg/mL were obtained (Figure 3). These strains have piqued our interest as they are classified as being susceptible to meropenem according to the EUCAST guidelines [22]. It has been recommended that carbapenems be used as monotherapy for treating patients with infections caused by such strains. Therefore, the ability of bacteria to produce carbapenemase enzymes is not taken into account, and susceptibility to meropenem is given the highest priority. Considering this, we decided to investigate how a transconjugant strain that is susceptible to meropenem and produces carbapenemases might behave under meropenem exposure in an in vitro dynamic model (HFIM) that simulates its clinical dosing regimen. Will there be a loss of plasmid, or, conversely, will the ability to resist meropenem improve?
Therefore, in the second part of our study, we conducted a pharmacodynamic evaluation of meropenem using a hollow-fiber infection model and a KPC carbapenemase-producing transconjugant strain E. coli C600/565 with a meropenem MIC of 2 µg/mL. We simulated the pharmacokinetic profile observed in epithelial lining fluid following a high dose of meropenem in the hollow-fiber infection model (HFIM), in order to mimic a lung infection scenario. We obtained a series of kinetic time–kill curves that display the course of both the total and resistant bacterial populations for the tested strain. As seen in Figure 5, the ability of an E. coli C600/565 strain to produce carbapenemases determines its survival capability during antibiotic exposure as it exhibited extremely intensive growth under meropenem exposure that was not the case with the recipient strain E. coli C600. Similar results were obtained in our previous study [40]. To be honest, bacterial resistance to carbapenems in in vitro pharmacodynamic experiments develops slowly or does not develop at all, unless it is caused by the production of carbapenemases [41,42]. Possibly, under the meropenem exposure there appears to be further stabilization of plasmids within cells. This may especially be the case, as the presence of plasmids in cells provides an advantage to their survival. In addition, we assume that intensive growth of resistant cells and the lack of response to meropenem in E. coli C600/565 can also be attributed to the upregulation of carbapenemase genes located on the plasmid induced by the antibiotic. This explanation is supported by numerous previous reports of such phenomena in bacteria that are reflected in one of the published studies [31]. Additionally, we observed a similar pattern in the absence of a meropenem effect against the clinical strain of K. pneumoniae 1456 producing OXA-48 carbapenemase, which had an MIC of 2 μg/mL, similar to that of E. coli C600/565 [43]. In light of the above, it seems that the intensive development of resistance in both instances may be due to the upregulation of carbapenemase genes. Therefore, there is a hidden threat that initially highly susceptible, non-carbapenemase-producing organisms may acquire plasmids carrying carbapenemases genes and may not respond to meropenem therapy, since they can produce carbapenemases. To combat strains for which the effectiveness of meropenem may be significantly reduced, it is essential to explore preventive strategies. The WHO supports countries in reducing antimicrobial resistance by strengthening infection prevention and control measures, such as ensuring effective sanitation and hygiene across all healthcare settings [44]. In addition, a strategy involving shorter durations of antibiotic treatment is relevant at this time, as clinical trials have shown that it minimizes the risk of bacterial resistance to antibiotics [45]. This is due to the fact that, in addition to resistance arising from the production of carbapenemases, clinical strains can also develop porin mutations and activate efflux pumps during prolonged antibiotic treatments [46,47]. In turn, these mechanisms confer cross-resistance to other antibiotics, whether they belong to the same class or not. For example, overexpression of MexAB-OprM confers resistance to quinolones and most beta-lactam antibiotics (including meropenem) [48].
Our study has several limitations. It did not include a large number of strains with a wide range of MICs or other carbapenemase types, for example, metallo-beta-lactamases. Moreover, a more in-depth investigation using genetic methods could also help answer some of the questions raised during the study. Particularly, we did not determine the plasmid copy number in donor strains that is one of the possible factors that may influence their efficacy and therefore conjugation frequency.

5. Conclusions

The present study identified several key findings. Firstly, the frequency of conjugation between K. pneumoniae and either K. pneumoniae or E. coli was positively related with meropenem susceptibility of the K. pneumoniae donor strain. Secondly, in both recipient strains of K. pneumoniae, and E. coli, the acquisition of plasmids containing blaKPC carbapenemase genes resulted in higher MICs of meropenem compared to those containing blaOXA-48 carbapenemase genes. Thirdly, the frequency of conjugation between K. pneumoniae and E. coli falls within similar ranges. Although among the E. coli colonies isolated in mating experiments, only approximately 25% accepted plasmids compared to 100% in K. pneumoniae. This indicates a higher permissiveness of K. pneumoniae for plasmids from donor strains of K. pneumoniae, i.e., from bacteria of the same species. Fourthly, conjugation did not occur between K. pneumoniae and P. aeruginosa, as colonies of P. aeruginosa growing on selective agar failed to maintain plasmids. At the same time, their MICs for meropenem increased compared to recipients (4–8 µg/mL vs 0.125–0.25 µg/mL), likely due to alternative resistance mechanisms. A resistance mechanism due to efflux pumps was detected in three of six isolates of P. aeruginosa. Finally, the transconjugants with plasmids carrying carbapenemase genes and meropenem MICs at the upper limit of the “susceptibility” range may pose a potential threat to the efficacy of meropenem in clinical settings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biomedicines13010238/s1, Table S1. List of PCR primer used for the experiments; Table S2. MIC data (in µg/mL) of recipients and selected transconjugants following in vitro mating with donor K. pneumoniae 565; Table S3. MIC data (in µg/mL) of recipients and selected transconjugants following in vitro mating with donor K. pneumoniae 485; Table S4. MIC data (in µg/mL) of recipients and selected transconjugants following in vitro mating with donor K. pneumoniae 38; Table S5. Conjugation frequencies between donor K. pneumoniae and recipient K. pneumoniae, E. coli and P. aeruginosa strains. All data are represented as arithmetic mean ± standard error of the mean; Figure S1. A schematic illustration of the hollow-fiber infection model (HFIM); Figure S2. Agarose gel electrophoresis of K. pneumoniae, E. coli and P. aeruginosa transconjugants amplification products, 10V/cm, 0.9% agarose, DNA size markers 250–10,000 bp or 100–1500 bp; Figure S3. “Possible” transconjugant colonies of P. aeruginosa grown on agar with rifampicin and meropenem.

Author Contributions

Conceptualization, M.V.G. and J.R.S.; methodology, M.V.G. and J.R.S.; validation, M.V.G., J.R.S. and D.A.K.; formal analysis, J.R.S.; investigation, M.V.G., J.R.S. and D.A.K.; data curation, M.V.G. and J.R.S.; writing—original draft preparation, M.V.G. and J.R.S.; writing—review and editing, D.A.K.; visualization, J.R.S. and D.A.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

We wish to acknowledge Ageevets A.V. of the Pediatric Research and Clinical Center for Infectious Diseases, Saint Petersburg, Russia, for providing the K. pneumoniae clinical isolates and E. coli C600 laboratory strain.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
HFIMHollow-fiber infection model
CFConjugation frequency
MICMinimum inhibitory concentration
LBLuria broth
LALuria agar
PCRPolymerase chain reaction

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Figure 1. Schematic representation of the conjugation protocol.
Figure 1. Schematic representation of the conjugation protocol.
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Figure 2. Schematic representation of the pharmacodynamic experiment protocol.
Figure 2. Schematic representation of the pharmacodynamic experiment protocol.
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Figure 3. Flowchart followed to perform bacterial mating in pairs K. pneumoniae–K. pneumoniae, K. pneumoniae–E. coli, and K. pneumoniae–P. aeruginosa, and meropenem susceptibility of transconjugant strains. “Plasmid+” or “plasmid−” indicates the confirmation or not of plasmids in isolates that were tested for meropenem susceptibility. Susceptibility breakpoints were based on EUCAST recommendations: ≤2 µg/mL—susceptible, >8 µg/mL—resistant [22].
Figure 3. Flowchart followed to perform bacterial mating in pairs K. pneumoniae–K. pneumoniae, K. pneumoniae–E. coli, and K. pneumoniae–P. aeruginosa, and meropenem susceptibility of transconjugant strains. “Plasmid+” or “plasmid−” indicates the confirmation or not of plasmids in isolates that were tested for meropenem susceptibility. Susceptibility breakpoints were based on EUCAST recommendations: ≤2 µg/mL—susceptible, >8 µg/mL—resistant [22].
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Figure 4. The PCR-based conjugation frequency between donor carbapenemase-producing K. pneumoniae strains and plasmid-free recipient strains of K. pneumoniae, E. coli, and P. aeruginosa. The data presented as arithmetic means ± standard deviations (n = 3).
Figure 4. The PCR-based conjugation frequency between donor carbapenemase-producing K. pneumoniae strains and plasmid-free recipient strains of K. pneumoniae, E. coli, and P. aeruginosa. The data presented as arithmetic means ± standard deviations (n = 3).
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Figure 5. Time courses of the total bacterial population (0 × MIC) and meropenem-resistant (2×, 4×, 8× and 16 × MIC) sub-populations of recipient (a) and transconjugant (b) carbapenemase-producing strain of E. coli in pharmacodynamic experiments. Arrows indicate the start of the meropenem infusion. The data presented as arithmetic means± standard deviation (n = 3). Error bars represent standard deviation.
Figure 5. Time courses of the total bacterial population (0 × MIC) and meropenem-resistant (2×, 4×, 8× and 16 × MIC) sub-populations of recipient (a) and transconjugant (b) carbapenemase-producing strain of E. coli in pharmacodynamic experiments. Arrows indicate the start of the meropenem infusion. The data presented as arithmetic means± standard deviation (n = 3). Error bars represent standard deviation.
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Table 1. General characteristics of donor K. pneumoniae strains and their plasmids.
Table 1. General characteristics of donor K. pneumoniae strains and their plasmids.
K. pneumoniae Strain Number38485565
City, yearMoscow, 2011Saint Petersburg, 2012Saint Petersburg, 2012
PlasmidpOXAAPSS2pOXAAPSS1pKPCAPSS
NCBI reference sequenceNZ_KU159086.1NZ_KU159085.1NZ_KP008371.1
Incompatibility groupIncLIncLIncFII
Length63,359 bp66,284 bp127,970 bp
ResistomeblaOXA-48blaOXA-48, blaTEM-1bblaKPC-2, blaTEM-1b, qnrS1, mphA, mrx, mphR
ST of donorST147ST395ST273
MIC of meropenem, µg/mL163264
ST—sequence type; MIC—minimum inhibitory concentration.
Table 2. MICs of meropenem against tested bacterial strains.
Table 2. MICs of meropenem against tested bacterial strains.
Bacterial StrainCarbapenemaseMeropenem MIC, µg/mL
Klebsiella pneumoniae 38OXA-4816
Klebsiella pneumoniae 485OXA-4832
Klebsiella pneumoniae 565KPC64
Klebsiella pneumoniae ATCC 700603 1None0.06
Klebsiella pneumoniae 188 1None0.03
Escherichia coli ATCC 25922 1None0.03
Escherichia coli C600 1None0.03
Pseudomonas aeruginosa ATCC 9027 1None0.125
Pseudomonas aeruginosa ATCC 27853 1None0.25
1—rifampicin-resistant mutants.
Table 3. MICs of meropenem in the presence and absence of efflux pump inhibitor CCCP against tested P. aeruginosa isolates from matings with K. pneumoniae.
Table 3. MICs of meropenem in the presence and absence of efflux pump inhibitor CCCP against tested P. aeruginosa isolates from matings with K. pneumoniae.
P. aeruginosa IsolateMeropenem MIC, µg/mLMeropenem MIC in the Presence of CCCP, µg/mL
565/90271616
565/2785340.125
485/9027168
485/27853161
38/902740.125
38/2785388
In bold are highlighted P. aeruginosa strains, for which efflux has been confirmed through a 4-fold or higher decrease in meropenem MICs in the presence of CCCP.
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Savelieva, J.R.; Kondratieva, D.A.; Golikova, M.V. Intra- and Interspecies Conjugal Transfer of Plasmids in Gram-Negative Bacteria. Biomedicines 2025, 13, 238. https://doi.org/10.3390/biomedicines13010238

AMA Style

Savelieva JR, Kondratieva DA, Golikova MV. Intra- and Interspecies Conjugal Transfer of Plasmids in Gram-Negative Bacteria. Biomedicines. 2025; 13(1):238. https://doi.org/10.3390/biomedicines13010238

Chicago/Turabian Style

Savelieva, Julia R., Daria A. Kondratieva, and Maria V. Golikova. 2025. "Intra- and Interspecies Conjugal Transfer of Plasmids in Gram-Negative Bacteria" Biomedicines 13, no. 1: 238. https://doi.org/10.3390/biomedicines13010238

APA Style

Savelieva, J. R., Kondratieva, D. A., & Golikova, M. V. (2025). Intra- and Interspecies Conjugal Transfer of Plasmids in Gram-Negative Bacteria. Biomedicines, 13(1), 238. https://doi.org/10.3390/biomedicines13010238

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