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Communication

A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes

by
María Getino
1,*,†,
María López-Díaz
2,‡,
Nicholas Ellaby
2,
John Clark
3,
Matthew J. Ellington
2 and
Roberto M. La Ragione
1,4,*
1
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, University of Surrey, Guildford GU2 7AL, UK
2
UK Health Security Agency, Colindale, London NW9 5EQ, UK
3
Department of Microbiology, Epsom and St Helier University Hospitals NHS Trust, Carshalton SM5 1AA, UK
4
School of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK
*
Authors to whom correspondence should be addressed.
†
Current address: NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance, Department of Infectious Disease, Faculty of Medicine, Imperial College London, London W6 8RF, UK.
‡
Current address: Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), 15006 A Coruña, Spain.
Antibiotics 2022, 11(11), 1641; https://doi.org/10.3390/antibiotics11111641
Submission received: 1 November 2022 / Revised: 11 November 2022 / Accepted: 15 November 2022 / Published: 17 November 2022
(This article belongs to the Special Issue Advances in Plasmid Mediated Antimicrobial Resistance)
Browse Figures
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Abstract

:
IncL/M broad-host-range conjugative plasmids are involved in the global spread of blaOXA-48 and the emergence of blaNDM-1. The aim of this study was to evaluate the transmission potential of plasmids encoding the emergent NDM-1 carbapenemase compared to the pandemic OXA-48. The conjugation rate and fitness cost of IncM2 and IncL plasmids encoding these carbapenemase genes were tested using a variety of host bacteria. Genomic analysis of uropathogenic Escherichia coli SAP1756 revealed that blaNDM-1 was encoded on an IncM2 plasmid, which also harboured blaTEM-1, bleMBL and sul1 and was highly similar to plasmids isolated from the same geographical area. Conjugation experiments demonstrated that NDM-1 and OXA-48-carrying plasmids transfer successfully between different Enterobacterales species, both in vitro and in vivo. Interestingly, E. coli isolates tested as recipients belonging to phylogroups A, B1, D and F were able to receive IncM2 plasmid pSAP1756, while phylogroups B2, C, E and G were not permissive to its acquisition. In general, the IncL OXA-48-carrying plasmids tested transferred at higher rates than IncM2 harbouring NDM-1 and imposed a lower burden to their host, possibly due to the inactivation of the tir fertility inhibition gene and reflecting their worldwide dissemination. IncM2 plasmids carrying blaNDM-1 are considered emergent threats that need continuous monitoring. In addition to sequencing efforts, phenotypic analysis of conjugation rates and fitness cost are effective methods for estimating the pandemic potential of antimicrobial resistance plasmids.

1. Introduction

Carbapenemase-producing Enterobacterales (CPE) are classified as critical priority pathogens by the World Health Organization (WHO) [1]. Carbapenemases are acquired β-lactamases that prevent carbapenems, antibiotics used in healthcare settings as last resort, from effectively clearing bacterial infections caused by CPE. Apart from carbapenems, these enzymes are capable of hydrolysing penicillins, cephalosporins and monobactams. They are classified in class A, B or D, depending on the composition of their active site (serine for classes A and D and divalent cations for class B) [2]. Class B β-lactamases, including metallo-β-lactamases, hydrolyse a broad variety of β-lactams (except monobactams) in a zinc-dependent manner, while Class D β-lactamases, including OXA-48-like carbapenemases, produce a relatively weak hydrolysis of penicillins and carbapenems, but not cephalosporins, which makes them more difficult to detect [3]. These enzymes are frequently encoded on mobile genetic elements such as conjugative plasmids along with numerous other antimicrobial resistance (AMR) genes [4].
Conjugative plasmids are considered major vehicles in the spread of AMR [5]. They are capable of horizontally transferring between bacteria, ensuring their stability through toxin-antitoxin and partitioning systems, as well as counteracting their own fitness cost via mutation. These mechanisms allow conjugative plasmids to successfully spread and persist, even in the absence of antibiotics. Broad-host-range conjugative plasmids are of special high risk as they enable the transfer of multiple AMR genes between different bacterial species.
An example of broad-host-range plasmids involved in the spread of AMR genes is the IncL/M incompatibility group, responsible for the worldwide dissemination of the blaOXA-48 carbapenemase gene [6]. OXA-48 is a class D β-lactamase first isolated from a K. pneumoniae resistant to all β-lactams in Turkey in 2001 [7]. The blaOXA-48 gene, frequently carried by a conserved and highly transmissible plasmid backbone, was further disseminated to become one of the most prevalent carbapenemases in Europe, Northern Africa and the Middle East [8].
Although IncL/M plasmids are known for the spread of OXA-48, they have also been connected with the emergence of NDM-1 carbapenemase [9,10]. NDM-1, or New Delhi metallo-β-lactamase, is a class B1 β-lactamase first discovered in 2009 in a K. pneumoniae clinical isolate from a Swedish patient who had travelled to New Delhi, India [11]. Since then, it has been reported in numerous Gram-negative bacterial species and transmissible plasmids around the world, including IncN, IncA/C, IncF, IncH and IncL/M incompatibility groups [12]. In particular, a recent study performed in the UK, which analysed the sequence of 44 NDM-1-positive isolates belonging to five different bacterial species, suggested that the blaNDM-1 gene has been recently acquired on multiple occasions by IncL/M plasmids in geographically widespread isolates [13].
IncL/M plasmids were historically classified within the same incompatibility group [14]. However, these plasmids have been re-classified into IncL and IncM groups, compatible with each other and therefore belonging to two different incompatibility groups, with an overall nucleotide identity of approximately 94% [15]. IncL plasmids frequently encode OXA-48, whereas NDM-1 tend to appear in the IncM2 group [15]. One of the key differences observed between these two groups of plasmids is the inactivation of the fertility inhibition gene tir in IncL plasmids, which increases their conjugation rate facilitating the dissemination of the gene encoding OXA-48 [16].
In this study, a panel of clinical Enterobacterales isolates harbouring IncL or IncM2 plasmids, encoding OXA-48 and NDM-1, respectively, was phenotypically characterised using conjugation and fitness studies with the aim of evaluating the role of different bacterial hosts and plasmid backbones in the transmission potential of emergent and pandemic carbapenemase genes.

2. Results and Discussion

2.1. Uropathogenic Escherichia coli SAP1756 Harbours an IncM2 Plasmid Encoding blaNDM-1

Whole genome sequencing and genomic analysis of the uropathogenic E. coli SAP1756 confirmed the species was E. coli and belongs to ST130. Plasmid analysis identified an IncL/M conjugative plasmid carrying four AMR genes: blaNDM-1, blaTEM-1, bleMBL and sul1. MOBsuite tool allowed the reconstruction of the plasmid pSAP1756 from Illumina short reads and the sequence was compared to closely related publicly available plasmids identified by BLASTn (Figure 1).
One of the most similar plasmids in terms of size and gene content to pSAP1756 (red ring in Figure 1) found in Genbank was pEsST2350_SE_NDM (blue ring in Figure 1). Interestingly, this plasmid was found in another E. coli isolate of different sequence type (ST2350) collected from a human screening rectal sample, also in a South-East England hospital in 2018. A nearly identical plasmid, pKp_SE1_NDM (accession number: CM010662), was identified in K. pneumoniae (ST534) isolated from a human rectal sample in the same hospital in 2017 [13], suggesting that this specific plasmid variant could have been transmitting locally between different Enterobacterales species between 2017 and 2018.
Figure 1 also shows that all the AMR genes, including blaTEM-1, aacC2, blaNDM-1, blaDHA-1, sul1 and mph2, are clustered in the most variable region of these plasmids. The presence of genes typical in transposons and insertion elements in this variable region (e.g., tnpA, tnpU, tnpD, iscr and insL) reflects the ability of these plasmids to adapt to different antibiotics through the integration of mobile genetic elements carrying different AMR genes. On the other hand, plasmid genes involved in essential plasmid functions, including conjugation (e.g., tra genes, trb genes, mobC), replication (rep genes), and stability (e.g., pemK/pemI toxin-antitoxin system and parAB partitioning system), were grouped in the most conserved region, the plasmid backbone.

2.2. IncL OXA-48-Carrying Plasmids Transfer at Higher Rates than IncM2 Harbouring NDM-1

In a previous study, clinical isolates of five different species (E. coli, Enterobacter cloacae, Citrobacter sp., K. pneumoniae and Klebsiella oxytoca) isolated across the UK were found to harbour closely related IncM2 plasmids carrying blaNDM-1, suggesting inter-species horizontal gene transfer [13]. Representative isolates from that study were included here to quantify the threat posed by these emergent NDM-1 plasmids in comparison with the globally disseminated OXA-48 plasmids.
To test their ability to transfer between common bacterial pathogens, clinical isolates from different species were used as donors in conjugation experiments, with E. coli MG1655 or J53 as recipients. Two subtypes of IncL/M plasmids were compared, carrying blaNDM-1 (IncM2 plasmids) or blaOXA-48 (IncL plasmids).
Conjugation of IncM2 plasmids encoding NDM-1 was successful in five out of six cases, while three out of five IncL plasmids carrying OXA-48 were able to conjugate from the original donors (Figure 2A). The donors E. coli NDM-1_5, K. pneumoniae OXA-48_2 and Citrobacter OXA-48_4, were unable to produce transconjugants in the tested conditions and were excluded from the results graph. The reason for this inability to transfer could potentially be related to fertility inhibition systems present in co-resident plasmids of the donor bacteria [18].
Conjugation frequencies (CF) of those plasmids able to transfer were highly diverse, ranging between 10−4 and 10−1 for IncM2 NDM-1-carrying plasmids and between 10−2 and 1 for IncL OXA-48 plasmids. K. pneumoniae pNDM-MAR and K. pneumoniae pNDM-KN were included as examples of alternative plasmid types carrying the blaNDM-1 gene (IncH and IncA/C, respectively), and their transfer frequencies were in the lower range compared to the IncM2 and IncL plasmids tested.
When the influence of the donor background was discarded by using E. coli MG1655 as the donor (Figure 2B), all IncM2 NDM-1-harbouring plasmids conjugated at homogeneous frequencies (between 10−2 and 10−3), highlighting the similarities between their plasmid backbone [13]. pCTX-M-3 plasmid was added as a control IncM2 plasmid with a different cargo (blaCTX-M-3) and its conjugation frequency was ~10−2. Interestingly, the three IncL OXA-48 plasmids tested in these conditions transferred at a higher rate than IncM2 plasmids, ranging between 10−2 and 10−1.
As a general rule, the IncL OXA-48-carrying plasmids are able to transfer at a slightly higher rate than IncM2 NDM-1 plasmids, a result consistent with previous studies and possibly due to the inactivation of the fertility inhibition gene tir in IncL plasmids [16]. However, most NDM-1 plasmids tested here were able to successfully conjugate from the original donor and the newly formed transconjugants, highlighting the importance of monitoring these emergent plasmids as potential sources of antibiotic-resistant outbreaks.

2.3. Most E. coli Isolates Tested as Recipients Were Not Permissive to pSAP1756 Entry

A panel of 11 pathogenic and commensal E. coli isolates belonging to 8 different phylogroups were selected to represent the common phylogroups and tested as recipients of pSAP1756 from the original E. coli donor, which belongs to phylogroup D. Some of these isolates were previously characterised in the lab by whole-genome sequencing [19] and their main features and transfer results are summarised in Table 1.
Conjugation was detected in isolates belonging to phylogroups A, B1, D and F, while no transfer was detected to those belonging to phylogroups B2, C, E and G. None of the recipients tested had resident IncL/M plasmids, so no entry exclusion was expected. Interestingly, the three B2 isolates, which are common human pathogens, were unable to receive pSAP1756, suggesting that B2 strains have mechanisms preventing the entry of these plasmids (e.g., restriction-modification, CRISPR-Cas). In future studies, it would be useful to test a higher number of sequenced recipients belonging to different phylogroups to find E. coli common mechanisms preventing plasmid acquisition and also to analyse the genomes of E. coli isolates carrying IncM2 NDM-1 plasmids to confirm the most common phylogroups carrying them as well as to understand the consequences of this potential bias.

2.4. pSAP1756 Is Capable of Conjugating to (and From) Different Species of Enterobacterales

E. coli has been used as a recipient in the previous conjugation experiments as it is one of the most important bacterial pathogens transmitting AMR genes. However, IncM2 plasmids are known to be broad-host-range and have been found in a wide range of bacterial hosts. To test their ability to transfer between different species, the studies used four type strains of Enterobacterales (belonging to the following species: S. marcescens, K. pneumoniae, E. cloacae and Y. enterocolitica) as donors and recipients of pSAP1756. Conjugation was successful in all cases, with frequencies ranging between 10−2 and 10−4 (Table 2).

2.5. pSAP1756 and pOXA-48_1 Transfer at Similar Rates in a Galleria Mellonella Model

To compare the transfer potential of plasmids harbouring NDM-1 and OXA-48 in vivo, representative examples (pSAP1756 and pOXA-48_1) were transferred to E. coli MG1655 and used as donors in Galleria mellonella conjugation experiments. These experiments were performed over a 4 h period following injection of 107 CFU of donors and recipients separately into Galleria larvae. Conjugation frequencies observed (~10−2) resembled those in vitro, but with no statistically significant differences found between both plasmids (Figure 3).

2.6. NDM-1-Harbouring Plasmids Impose a Higher Fitness Cost for the Growth of Their Host Than Those Carrying OXA-48

In addition to horizontal gene transfer ability, fitness cost is an important parameter to test the pandemic potential of a plasmid. Growth curves were performed for E. coli MG1655 in the presence and absence of IncM2 and IncL plasmids carrying carbapenemase genes to estimate the burden they impose in this host. In most cases, the tested plasmids had an associated fitness cost to the host when compared to the growth rate of the same bacteria with no plasmid (Figure 4). In addition, the burden associated with OXA-48 plasmids in E. coli MG1655 was slightly lower than the corresponding cost of NDM-1 plasmids, which could also contribute to the increased success of OXA-48 plasmids.

3. Materials and Methods

3.1. Bacterial Cultures, DNA Extraction and Genome Sequencing

A list of the bacterial isolates used in this study can be found in Table 3. E. coli SAP1756 was isolated from a human urinary tract infection source in the diagnostics laboratory of a South-East England hospital in 2017 and transferred to the University of Surrey for further analysis. The isolate was aseptically cultured on MacConkey agar No3 (Oxoid, Basingstoke, UK) plates for 18–24 h at 37 °C, aerobically. A single colony was then selected for storage at −80 °C using the Microbank preservation system (Pro-Lab Diagnostics, Bromborough, UK). Bacteria stored at −80 °C were aseptically inoculated into LB broth prior to culture for 18–24 h at 37 °C, aerobically with agitation at 200 rpm. DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Manchester, UK) following the manufacturer’s instructions. DNA quality and quantity were confirmed using Nanodrop and DNA was shipped to the Animal and Plant Health Agency (APHA, Weybridge, UK) for whole genome sequencing using an Illumina platform.

3.2. Genomic Analysis

Paired-end reads were quality-checked with FastQC v0.11.9 [25] and used for genome assembly through Spades v3.15.2 [26]. The quality of the SAP1756 assembly was determined with Quast v5.0.2 [27], and Kraken v2.1.2 [28]. MLST v2.19.0 allowed the identification of the Multi-Locus Sequence Type (ST) [29,30]. ABRicate v1.0.1 in combination with NCBI AMRfinderPlus v2021-Mar-27 and PlasmidFinder v2021-Mar-27 databases were used to detect AMR genes and plasmid replicons, respectively [31,32,33]. IncL/M plasmid was reconstructed with MOBsuite v3.0.0 [34], and used as a query for NCBI BLAST Nucleotide [35]. Closely related publicly available plasmid sequences were downloaded from NCBI and compared with SAP1756 genome assembly using BLAST Ring Image Generator [17].

3.3. In Vitro Conjugation Assays

Donors and recipients (Table 3) were cultured on LB broth supplemented with antibiotics for 18–24 h at 37 °C, aerobically with agitation at 200 rpm. The cultures were then centrifuged for 5 min at 8000 rpm at room temperature, the supernatant was discarded, and the pellet was resuspended in sterile LB broth. OD600 for each culture was measured and adjusted to 4. For each individual conjugation assay, 100 µL of donor and 100 µL of recipient bacteria were mixed prior to centrifugation for 5 min at room temperature at 8000 rpm. The supernatant was discarded, the pellet resuspended in 15 µL LB broth and the final mixture of concentrated donors and recipients was spotted onto a well of a 24-well plate previously filled with 1 mL LB agar and solidified. The mixture was spread on the surface of the LB agar to create a thin layer and allowed to dry at room temperature. Conjugation was then performed for 1 h at 37 °C and stopped by resuspending the harvested mixture in 1 mL PBS 1x. Serial dilutions were prepared and 3 × 10 µL drops of each dilution were plated in LB agar with the appropriate antibiotics to select for donors, recipients and transconjugants (Table 3). Conjugation frequency was estimated by dividing the number of transconjugants (CFU/mL) by donors or recipients (CFU/mL) and the average of 3 or more independent experiments was represented in a logarithmic scale. When needed for selection purposes, spontaneous mutants of donor and recipient strains resistant to rifampicin or nalidixic acid were used, selected by growing an overnight culture of the sensitive isolate in LB agar plates supplemented with the corresponding antibiotic.

3.4. In Vivo Conjugation Assays

E. coli MG1655 resistant to nalidixic acid (carrying plasmids pSAP1756 or pOXA-48_1) and E. coli MG1655 resistant to rifampicin were used as donors and recipients, respectively. Donors and recipients were cultured on LB broth supplemented with antibiotics for 18–24 h at 37 °C, aerobically with agitation at 200 rpm. The cultures were centrifuged for 5 min at 8000 rpm at room temperature, the supernatant was discarded, and the pellet was resuspended in sterile PBS 1x. A volume of 10 µL of donors and 10 µL of recipients containing ~107 CFU were separately injected into the right and left prolegs of Galleria mellonella larvae (TruLarv, Biosystems Technology, Crediton, UK) using microliter syringes (Hamilton, Bonaduz, Switzerland). Conjugation was permitted for 4 h at 37 °C. Larvae were euthanised by decapitation and then homogenised in 200 µL of sterile PBS 1x with a Micro Tissue Homogenizer (BioMasherII, Fisher Scientific, Loughborough, UK). As previously described, serial dilutions were prepared and 3 x 10 µL drops of each dilution were plated in LB agar with the appropriate antibiotics to select for donors (meropenem 0.25 μg/mL), recipients (rifampicin 50 μg/mL) and transconjugants (rifampicin 50 μg/mL + meropenem 0.25 μg/mL). Conjugation frequency was estimated by dividing transconjugants (CFU/mL) by donors (CFU/mL). The results represented the average of at least 8 independent experiments on a logarithmic scale.

3.5. Fitness Cost Experiments

Bacterial isolates were cultured on LB broth supplemented with the appropriate antibiotics for 18–24 h at 37 °C, aerobically and with agitation at 200 rpm. Cultures were centrifuged for 5 min at 8000 rpm at room temperature and resuspended in M9 minimal media supplemented with 0.4% glucose, adjusting to an OD600 of 0.01 or 0.001. A volume of 100 µL of diluted cultures was added to 96-well microplates and incubated for 24 h at 37 °C, aerobically in a Spark 10M microplate reader. OD600 was measured every 10 min after 20 s of double orbital shaking at 1 mm of amplitude and 270 rpm of frequency. The absorbance results (Ln2) were plotted over time and the generation time was estimated from the exponential phase of the graph by dividing Ln2 by the slope of the linear trendline. At least 5 growth curves were used to calculate the average generation time of a given isolate. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test comparing each plasmid-carrying bacterial host versus the control (host without plasmid) was performed using GraphPad Prism.

4. Conclusions

The studies reported here demonstrated that IncL/M plasmids are vehicles of blaNDM-1 and blaOXA-48 genes able to disseminate these carbapenemase genes to a broad number of bacterial hosts. In general, the emergent IncM2 NDM-1-harbouring plasmids tested under the conditions of this study had a lower transfer rate and higher fitness cost when compared with the pandemic IncL OXA-48-carrying plasmids, a potential cause for the more extensive global spread of OXA-48 plasmids. However, it is important to continue monitoring plasmids carrying carbapenemase and other resistance genes that could be a threat to human and animal health due to the rapid evolution and adaptability of mobile genetic elements.

Author Contributions

Conceptualisation, M.G., M.J.E. and R.M.L.R.; methodology, M.G.; software, M.G.; validation, M.G.; formal analysis, M.G.; investigation, M.G., M.L.-D., N.E., J.C., M.J.E. and R.M.L.R.; resources, M.J.E. and R.M.L.R.; data curation, M.G.; writing—original draft preparation, M.G.; writing—review and editing, M.G., M.L.-D., N.E., M.J.E. and R.M.L.R.; visualisation, M.G.; supervision, M.J.E. and R.M.L.R.; project administration, M.G., M.J.E. and R.M.L.R.; funding acquisition, M.J.E. and R.M.L.R. All authors have read and agreed to the published version of the manuscript.

Funding

This work was undertaken as part of the ARDIG project in the One Health European Joint Programme Consortium. This project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 773830.

Institutional Review Board Statement

A study protocol to collect anonymised patient data linked to the bacterial isolates provided was approved by the Health Research Authority under reference 19/HRA/0552. Capacity and capability were confirmed by the Research and Development department of the NHS site involved.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB57396 (https://www.ebi.ac.uk/ena/browser/view/PRJEB57396, accessed on 16 November 2022).

Acknowledgments

The authors thank the hospital microbiology staff and the veterinary practice staff for assisting with the collection of bacterial isolates.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Antibiotics 11 01641 g001 550
Figure 1. Comparison of closely related IncL/M plasmids, aligned against the reference plasmid pNDM-HK, using BLAST Ring Image Generator (BRIG) [17]. Genbank accession numbers, bacterial hosts, plasmid sizes and ring characteristics: pNDM-HK (HQ451074, E. coli, 88,803 bp, innermost ring, reference), pNDM-OM (JX988621, Klebsiella pneumoniae, 87,185 bp, purple ring), pIT-Sma-01/2014 (MH722219, Serratia marcescens, 88,129 bp, green ring), pIT-Cfr-01/2015 (MH722216, Citrobacter freundii, 78,923 bp, yellow ring), pEsST2350_SE_NDM (CP031322, E. coli, 75,645 bp, blue ring) and pSAP1756 ((E. coli, 73,743 bp, red ring). The outermost ring shows the genes identified in the reference plasmid pNDM-HK and the diagram next to the innermost ring represents the GC content of the reference plasmid.
Figure 1. Comparison of closely related IncL/M plasmids, aligned against the reference plasmid pNDM-HK, using BLAST Ring Image Generator (BRIG) [17]. Genbank accession numbers, bacterial hosts, plasmid sizes and ring characteristics: pNDM-HK (HQ451074, E. coli, 88,803 bp, innermost ring, reference), pNDM-OM (JX988621, Klebsiella pneumoniae, 87,185 bp, purple ring), pIT-Sma-01/2014 (MH722219, Serratia marcescens, 88,129 bp, green ring), pIT-Cfr-01/2015 (MH722216, Citrobacter freundii, 78,923 bp, yellow ring), pEsST2350_SE_NDM (CP031322, E. coli, 75,645 bp, blue ring) and pSAP1756 ((E. coli, 73,743 bp, red ring). The outermost ring shows the genes identified in the reference plasmid pNDM-HK and the diagram next to the innermost ring represents the GC content of the reference plasmid.
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Antibiotics 11 01641 g002 550
Figure 2. Conjugation frequency (CF) of IncL/M plasmids harbouring blaNDM-1 or blaOXA-48 genes, represented as the logarithm of transconjugants per donor obtained after 1 h conjugation at 37 °C on LB agar. (A) Conjugation from the original donors to E. coli MG1655 (rifampicin or nalidixic acid spontaneous mutants), or J53 in cases where donors were resistant to both rifampicin and nalidixic acid. Plasmids encoding NDM-1 but belonging to other incompatibility groups were added for comparison (pNDM-MAR and pNDM-KN, IncH and IncA/C, respectively). (B) Conjugation from the transconjugants from A in E. coli MG1655 (spontaneous mutant resistant to nalidixic acid) to E. coli MG1655 (spontaneous mutant resistant to rifampicin). Circles represent the result of individual experiments with NDM-1-carrying IncM2 plasmids (except control plasmid pCTX-M-3, which is NDM-1-negative) and squares represent CF with IncL plasmids encoding OXA-48. Horizontal lines show the mean and vertical lines the standard deviation of at least 4 independent experiments.
Figure 2. Conjugation frequency (CF) of IncL/M plasmids harbouring blaNDM-1 or blaOXA-48 genes, represented as the logarithm of transconjugants per donor obtained after 1 h conjugation at 37 °C on LB agar. (A) Conjugation from the original donors to E. coli MG1655 (rifampicin or nalidixic acid spontaneous mutants), or J53 in cases where donors were resistant to both rifampicin and nalidixic acid. Plasmids encoding NDM-1 but belonging to other incompatibility groups were added for comparison (pNDM-MAR and pNDM-KN, IncH and IncA/C, respectively). (B) Conjugation from the transconjugants from A in E. coli MG1655 (spontaneous mutant resistant to nalidixic acid) to E. coli MG1655 (spontaneous mutant resistant to rifampicin). Circles represent the result of individual experiments with NDM-1-carrying IncM2 plasmids (except control plasmid pCTX-M-3, which is NDM-1-negative) and squares represent CF with IncL plasmids encoding OXA-48. Horizontal lines show the mean and vertical lines the standard deviation of at least 4 independent experiments.
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Antibiotics 11 01641 g003 550
Figure 3. Conjugation frequency (CF) of IncM2 NDM-1-carrying plasmid pSAP1756 and IncL OXA-48-carrying plasmid pOXA48_1 using E. coli MG1655 resistant to nalidixic acid as donor and E. coli MG1655 resistant to rifampicin as recipient. Blue circles and orange squares represent the logarithm of transconjugants per donor obtained per experiment after 4 h conjugation in Galleria mellonella inoculated with ~107 CFU of donors and recipients separately. Horizontal lines show the mean and vertical lines the standard deviation of at least 8 independent experiments.
Figure 3. Conjugation frequency (CF) of IncM2 NDM-1-carrying plasmid pSAP1756 and IncL OXA-48-carrying plasmid pOXA48_1 using E. coli MG1655 resistant to nalidixic acid as donor and E. coli MG1655 resistant to rifampicin as recipient. Blue circles and orange squares represent the logarithm of transconjugants per donor obtained per experiment after 4 h conjugation in Galleria mellonella inoculated with ~107 CFU of donors and recipients separately. Horizontal lines show the mean and vertical lines the standard deviation of at least 8 independent experiments.
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Antibiotics 11 01641 g004 550
Figure 4. Generation time of E. coli MG1655 (resistant to nalidixic acid) in hours, with (orange circles) or without (blue squares) IncL/M plasmids, calculated during the exponential phase of growth in M9 minimal media supplemented with glucose. The differential growth of the host without and with plasmid represents the burden of the plasmid under these conditions. Horizontal lines show the mean and vertical lines the standard deviation of at least 5 independent experiments. One-way analysis of variance with Dunnett’s post hoc test was performed between the bacterial hosts with plasmid versus the control without plasmid (*: p < 0.05; ns: no significant difference).
Figure 4. Generation time of E. coli MG1655 (resistant to nalidixic acid) in hours, with (orange circles) or without (blue squares) IncL/M plasmids, calculated during the exponential phase of growth in M9 minimal media supplemented with glucose. The differential growth of the host without and with plasmid represents the burden of the plasmid under these conditions. Horizontal lines show the mean and vertical lines the standard deviation of at least 5 independent experiments. One-way analysis of variance with Dunnett’s post hoc test was performed between the bacterial hosts with plasmid versus the control without plasmid (*: p < 0.05; ns: no significant difference).
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Table
Table 1. Pathogenic and commensal E. coli isolates used as recipients in conjugation experiments.
Table 1. Pathogenic and commensal E. coli isolates used as recipients in conjugation experiments.
E. coli RecipientTypePhylogroupSourceYearCountryCF (log10 T/R)
MG1655 (RifR)CommensalAHuman1922US−2.1 ± 0.2
SAP2531 (RifR)PathogenicAHuman2018UK−3.8 ± 0.7
SAP2592 (RifR)PathogenicB1Human2018UK−3.4 ± 0.6
NCTC 12241 (RifR)PathogenicB2Human1946USN.D.
NCTC 13441 (NxR)PathogenicB2Human2003UKN.D.
SAP2492 (RifR)PathogenicB2Human2018UKN.D.
SAP2188 (NxR)PathogenicCAvian2018UKN.D.
SAP2526 (NxR)PathogenicDHuman2018UK−4.4 ± 0.4
SAP2573 (RifR)PathogenicEHuman2018UKN.D.
SAP2485 (RifR)PathogenicFHuman2018UK−3.6 ± 0.2
SAP2180 (RifR)PathogenicGAvian2018UKN.D.
CF: conjugation frequency (mean ± standard deviation) of IncM2 plasmid pSAP1756 using the original host as donor (SAP1756, phylogroup D). N.D.: no transconjugants were detected in one or more experiments. RifR: resistant to rifampicin. NxR: resistant to nalidixic acid. T: transconjugants/mL. R: recipients/mL.
Table
Table 2. Conjugation frequency of IncM2 plasmid pSAP1756 from the original host to different Enterobacterales species and from these different species to E. coli MG1655.
Table 2. Conjugation frequency of IncM2 plasmid pSAP1756 from the original host to different Enterobacterales species and from these different species to E. coli MG1655.
DonorPlasmidRecipientCF (log10 T/R)
E. coli SAP1756pSAP1756E. coli MG1655 (RifR)−2.1 ± 0.2
E. coli SAP1756pSAP1756S. marcescens NCTC 13382 (RifR)−3.0 ± 0.2
E. coli SAP1756pSAP1756K. pneumoniae NCTC 9633 (RifR)−3.6 ± 0.4
E. coli SAP1756pSAP1756E. cloacae NCTC 13380 (RifR)−4.0 ± 0.1
E. coli SAP1756pSAP1756Y. enterocolitica NCTC 12982 (RifR)−2.4 ± 0.3
DonorPlasmidRecipientCF (log10 T/D)
E. coli MG1655 (RifR)pSAP1756E. coli MG1655 (NxR)−2.4 ± 0.2
S. marcescens NCTC 13382 (RifR)pSAP1756E. coli MG1655 (NxR)−2.3 ± 0.4
K. pneumoniae NCTC 9633 (RifR)pSAP1756E. coli MG1655 (NxR)−2.5 ± 0.7
E. cloacae NCTC 13380 (RifR)pSAP1756E. coli MG1655 (NxR)−1.9 ± 0.2
Y. enterocolitica NCTC 12982 (RifR)pSAP1756E. coli MG1655 (NxR)−1.8 ± 0.5
CF: conjugation frequency (mean ± standard deviation); RifR: resistant to rifampicin; NxR: resistant to nalidixic acid; T: transconjugants/mL; D: donors/mL; R: recipients/mL.
Table
Table 3. Bacterial strains and plasmids used in this study.
Table 3. Bacterial strains and plasmids used in this study.
SpeciesIsolatePlasmidInc AMR GeneD/RSelectionReference
E. coliSAP1756pSAP1756IncM2blaNDM-1DMer0.25This work
E. coliSAP2180 RNx50This work
E. coliSAP2188 RNx50This work
E. coliSAP2485 RRif50This work
E. coliSAP2492 RRif50[19]
E. coliSAP2526 RNx50[19]
E. coliSAP2531 RRif50[19]
E. coliSAP2573 RRif50[19]
E. coliSAP2592 RNx50[19]
E. cloacaeNDM-1_1pNDM-1_1IncM2blaNDM-1DMer0.25[13]
CitrobacterNDM-1_2pNDM-1_2IncM2blaNDM-1DMer0.25[13]
K. pneumoniaeNDM-1_3pNDM-1_3IncM2blaNDM-1DMer0.25[13]
E. cloacaeNDM-1_4pNDM-1_4IncM2blaNDM-1DMer0.25[13]
E. coliNDM-1_5pNDM-1_5IncM2blaNDM-1DMer0.25[13]
E. coliOXA-48_1pOXA-48_1IncLblaOXA-48DMer0.25This work
K. pneumoniaeOXA-48_2pOXA-48_2IncLblaOXA-48DMer0.25This work
E. cloacaeOXA-48_3pOXA-48_3IncLblaOXA-48DMer0.25This work
CitrobacterOXA-48_4pOXA-48_4IncLblaOXA-48DMer0.25This work
K. oxytocaOXA-48_5pOXA-48_5IncLblaOXA-48DMer0.25This work
E. coliMG1655pCTX-M-3IncM2blaCTX-M-3DCtx2[20]
K. pneumoniaeTCKpnCpNDM-MARIncHblaNDM-1DMer0.25[21]
K. pneumoniaeKp7pNDM-KNIncA/CblaNDM-1DCtx2[22]
E. coliMG1655 RNx50/Rif50[23]
E. coliJ53 RNaN3100[24]
S. marcescensNCTC 13382 RRif50
K. pneumoniaeNCTC 9633 RRif50
E. cloacaeNCTC 13380 RRif50
Y. enterocoliticaNCTC 12982 RRif50
E. coliNCTC 12241 RRif50
E. coliNCTC 13441 RNx50
Inc: incompatibility group; D: donor; R: recipient; Mer0.25: meropenem 0.25 μg/mL; Nx50: nalidixic acid 50 μg/mL; Rif50: rifampicin 50 μg/mL; Ctx2: cefotaxime 2 μg/mL; NaN3100: sodium azide 100 μg/mL).
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Getino, M.; López-Díaz, M.; Ellaby, N.; Clark, J.; Ellington, M.J.; La Ragione, R.M. A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes. Antibiotics 2022, 11, 1641. https://doi.org/10.3390/antibiotics11111641

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Getino M, López-Díaz M, Ellaby N, Clark J, Ellington MJ, La Ragione RM. A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes. Antibiotics. 2022; 11(11):1641. https://doi.org/10.3390/antibiotics11111641

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Getino, María, María López-Díaz, Nicholas Ellaby, John Clark, Matthew J. Ellington, and Roberto M. La Ragione. 2022. "A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes" Antibiotics 11, no. 11: 1641. https://doi.org/10.3390/antibiotics11111641

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