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Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients

Abstract

Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterize the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a 2-yr period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonized patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.

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Fig. 1: Study population, colonized patients and pOXA-48-carrying enterobacteria.
Fig. 2: Phylogenetic analysis of pOXA-48-carrying K. pneumoniae and E. coli.
Fig. 3: SCOTTI reconstruction of between-patient transfer of pOXA-48-carrying enterobacteria.
Fig. 4: Within-patient pOXA-48 transfer.
Fig. 5: pOXA-48 conjugation rate.

Data availability

The sequences generated and analysed during the current study are available in the Sequence Read Archive (SRA) repository, BioProject ID: PRJNA626430. The closed, annotated pOXA-48 plasmids generated in this study are available under GenBank accession numbers MT441554 and MT989343MT989349. Source data are provided with this paper.

Code availability

The code generated during the current study is available in GitHub (http://www.github.com/leonsampedro/transmission_stan_code).

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Acknowledgements

This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (ERC grant agreement no. 757440-PLASREVOLUTION) and by the Instituto de Salud Carlos III (cofunded by the European Development Regional Fund (EDRF) ‘A way to achieve Europe’) grant nos. PI16-00860 and CP15-00012. The R-GNOSIS project received financial support from the European Commission (grant no. R-GNOSIS-FP7-HEALTH-F3-2011-282512). R.C. acknowledges financial support from the European Commission (R-GNOSIS) and Plan Nacional de I+D+i2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (grant no. REIPIRD16/0016/0011) cofinanced by EDRF ‘A way to achieve Europe’, Operative Programme Intelligent Growth 2014–2020. B.S.C. and T.C. acknowledge support from the UK Medical Research Council and Department for International Development (grant no. MR/K006924/1), and B.S.C. and P.M. acknowledge support under the framework of the JPIAMR (Joint Programming Initiative on Antimicrobial Resistance). A.S.M. is supported by a Miguel Servet Fellowship (grant no. MS15-00012). R.L.-S. thanks the Federation of European Microbiological Societies (FEMS) for a Research and Training Grant (no. FEMS-GO-2018-143). J.R.-B. is the recipient of a Juan de la Cierva-Incorporación Fellowship (grant no. IJC2018-035146-I) cofunded by Agencia Estatal de Investigación del Ministerio de Ciencia e Innovación. M.H.-G. was supported with a contract from Instituto de Salud Carlos III, Spain (iP-FIS programme, ref. IFI14/00022). We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of the sequencing data.

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Contributions

A.S.M., R.L.-S. and B.S.C. conceived the study. R.C. designed and supervised sampling and collection of bacterial isolates. M.H.-G. and P.R.-G. collected the bacterial isolates and performed bacterial characterization. C.D.-A. and N.L.-F. collected the epidemiological data and performed preliminary analyses. R-GNOSIS WP5 Study Group designed sampling protocols and facilitated the training and capacity building for the collection of bacterial isolates and preliminary analyses. J.D.F., J.R.-B. and C.d.l.V. performed the experimental work and analysed the results. R.L.-S., B.S.C., P.M. and T.C. performed the data analysis. A.S.M. coordinated the study. A.S.M. and R.L.-S. wrote the initial draft of the manuscript. A.S.M., R.L.-S., J.D.F., J.R.-B., B.S.C., P.M. and T.C. contributed to the final version of the manuscript. All authors read and approved the manuscript.

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Correspondence to Álvaro San Millán.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Amy Mathers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Plasmid pOXA-48.

a, Schematic representation of plasmid pOXA-48_K8 (GenBank Accession Number MT441554). The reading frames for genes are shown as arrows, with the direction of transcription indicated by the arrowhead. Arrow colours indicate the functional classification of the gene (see legend). The blaOXA-48 gene is indicated in pink. The IS1999.2 is highlighted in yellow. The direct repeats flanking the Tn1999.2 are indicated (CGTTCAGCA). pOXA-48_K8 carries a group II intron (indicated by ltrA) downstream pemK. This intron has also been detected in other pOXA-48-like plasmids, like pOXA48-L121 (ref. 64). SNPs positions detected in the pOXA-48 variants that were used to track within-patient transfer are indicated with triangles (green for synonymous SNPs and red for non-synonym SNPs, using pOXA48_K8 as reference). The position of the mutated nucleotides in the plasmid is indicated, as well as the aminoacidic changes for non-synonymous mutations in the proteins. The inner purple circle shows the core region of pOXA-48 variants that shared at least 60 kb (>90 % of the whole sequence, 219 out of 250, Supplementary Table 1), which were used to investigate within-patient plasmid transfer (Fig. 4). The numbers in the inner circle indicate the last/first nucleotide position of those regions included in the core sequence, using pOXA48_K8 as the reference. Most of the plasmids were sequenced with short-read technology, therefore the ISs are not included in the core-genome because they are repeated in the plasmid and do not map properly. The group II intron (indicated by ltrA) is not part of the core either because it is a highly promiscuous genetic element (see Supplementary Table 1 for details about presence/absence of this intron in the pOXA_48-like plasmids in our collection). Other genes excluded from the core regions are radC, korC and trbB. b, Comparison of pOXA-48-like plasmid sequences. Compared to the original pOXA-48 (ref. 14), pOXA-48_K8 carries the central part of Tn1999 inverted, with a copy of IS1 inserted in IS1999, upstream of blaOXA-48 (named Tn1999.2 (ref. 65)). Moreover, pOXA-48_K8 carries an additional copy of IS1 close to the korC gene, also found in plasmid pRA35 (ref. 66).

Extended Data Fig. 2 Patients sampled and patients colonised by pOXA-48-carrying enterobacteria over time.

a, Patients sampled during the R-GNOSIS study. Number of hospitalised patients in each ward over the 25-month study period, divided per month. b, Length of stay of patients per ward. Violin plots represent the distribution of length of stay per ward of all the patients admitted during the study period (neurosurgery, n = 1,068; gastroenterology, n = 2,591; pneumology, n = 2,559; urology, n = 3,483). Blue dots represent the length of stay of those patients colonised by pOXA-48-carrying enterobacteria (neurosurgery, n = 16; gastroenterology, n = 33; pneumology, n = 38; urology, n = 18). Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 times the interquartile range. c, Patients colonised by a pOXA-48-carrying enterobacteria during the study. Number of colonised patients in each ward, over the 25-month study period, divided per month (including every colonised patient, not only newly colonised patients). d, Distribution of days from admission to colonisation in each colonised patient divided per ward, represented as a boxplot (neurosurgery, n = 16; gastroenterology, n = 33; pneumology, n = 38; urology, n = 18). Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 times the interquartile range. e, Age of patients admitted in the different wards under study represented as boxplots and divided by sex. Lighter boxes represent the ages of all the patients admitted to the wards (neurosurgery: female= 498, male= 570; gastroenterology: female= 1,090, male= 1,501; pneumology: female= 1,131, male= 1,428; urology: female= 912, male= 2,571). Narrow darker boxes represent those patients colonised by a pOXA-48-carrying enterobacteria (neurosurgery: female= 7, male= 9; gastroenterology: female= 13, male= 20; pneumology: female= 18, male= 20; urology: female= 4, male= 14). Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 times the interquartile range.

Extended Data Fig. 3 Swab outcomes from patients colonised by pOXA-48-carrying enterobacteria.

Outcome of the samples recovered from the 105 patients colonised by a pOXA-48-carrying enterobacteria in the R-GNOSIS study. The figure shows the distribution of patients colonised by pOXA-48-carrying enterobacteria in the four wards under study over the 25-month study period. Each row represents a patient, and the black segments represent the length of hospital stay (from admission to discharge). Coloured points within the segments indicate the sampling dates. Red points indicate those samples from which a pOXA-48-carrying isolate was recovered (positive swabs), and green points indicate those samples from which no pOXA-48-carrying isolate was recovered (negative swabs). 29 of the 105 colonised patients were admitted in multiple occasions during the study period. The percentage of positive result for a pOXA-48-carrying enterobacteria in the first sample taken after a new admission of these 105 patients was 24.85% (41/165).

Extended Data Fig. 4 Acquisition of pOXA-48-carrying enterobacteria by hospitalised patients.

a, Posterior distribution of odds ratio for the daily risk of colonisation with a pOXA-48-carrying K. pneumoniae or E. coli (see methods for details). Two covariates were included. The first is the presence of other patients colonised by a pOXA-48-carrying clone on the ward, (upper part, stratified by ward). If between-patient transfer of the plasmid is important, we expect to see a positive association (odds ratio >1) with the daily probability of acquiring a pOXA-48 clone. Second, pre-existing colonisation with a pOXA-48 clone of a different species (lower part). This covariate measures how being previously colonised by a pOXA-48-carrying E. coli is associated with the daily probability of becoming colonized with a pOXA-48-carrying K. pneumoniae clone (Eco -> Kpn) and vice versa (Kpn -> Eco). We expect to see a positive association if within-patient transfer of pOXA-48 between different bacterial clones is important. Points represent posterior medians; thick grey lines represent the 80% credible interval (CrI) and thinner black lines represent the 95% CrI. b, Number of previously uncolonised patients becoming colonised by a pOXA-48-carrying K. pneumoniae (top row) or E. coli (bottom row) as a function of the number of patients on the ward already colonised by a pOXA-48-carrying clone. c, Number of R-GNOSIS study patients colonised by pOXA-48-carrying K. pneumoniae (Kpn) or E. coli (Eco) clones or both (co-colonised). For co-colonised patients, the colour code indicates whether K. pneumoniae or E. coli were isolated first or whether both species were simultaneously isolated from the same swab.

Extended Data Fig. 5 Phylogenetic analysis of isolates preliminary identified as K. pneumoniae.

Unrooted phylogeny of 108 whole genome assemblies from the clones phenotypically identified as K. pneumoniae. Branch length gives the mash distance (a measure of k-mer similarity) between assemblies. Note the three distinct clusters, which are considered to be separate species (distance > 0.05): K. pneumoniae (n = 103), K. quasipneumoniae (n = 2) and K. variicola (n = 3).

Extended Data Fig. 6 Spatiotemporal distribution of patients colonised by K. pneumoniae ST11 in the neurosurgery ward.

Distribution of patients colonised by pOXA-48-carrying K. pneumoniae ST11 in the neurosurgery ward. Each row represents a patient and the colour segments represent the length of stay in the hospital (from admission to discharge). The colours of the segments represent the different rooms within the ward (see legend). Arrows represent transmission events predicted by SCOTTI. Line thickness represents the probability of the transmission predicted by SCOTTI. The number to the right of the arrowhead indicates the number of SNPs between the complete core genomes of the pair of clones involved in the putative transmission event. Note that 6 out of 16 patients shared room G in overlapping stays.

Extended Data Fig. 7 pOXA-48-carrying enterobacteria analysed in this study.

Representation of the 250 pOXA-48-carrying clones isolated in the hospital from the first description till the end of the study period. The colour code indicates the species of the pOXA-48-carrying enterobacteria as indicated in the legend.

Extended Data Fig. 8 Conjugation frequency of plasmid pOXA-48.

Conjugation frequencies (transconjugants per donor) of the most common pOXA-48 variant in the hospital and the four variants with SNPs in the core region used to track within-patient plasmid transfer (n = 6 biological replicates). Conjugation experiments were performed on three different agar media: LB, MacConkey and M9 minimal medium supplemented with gluconate (MMG), and both in aerobic and anaerobic conditions. Plasmid variant numbers correspond to those indicated in Fig. 4. Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 times the interquartile range. The data presented here is the same as in Fig. 5, but represented as conjugation frequency instead of rate. Source data

Supplementary information

Supplementary Information

Supplementary Tables 2 and 3, Figs. 1–4 and the complete list of R-GNOSIS WP5 Study Group members.

Reporting Summary

Supplementary Table 1

Strains used in this study.

Supplementary Tables 2 and 3

Risk factors and the daily probability for acquisition of pOXA-48-carrying enterobacteria by hospitalized patients. Daily probability of acquisition of pOXA-48-carrying K. pneumoniae or E. coli among patients admitted to the four different wards during the study period of the R-GNOSIS project. (1) Probability of acquisition is the posterior intercept plus the posterior coefficients transformed onto the probability scale. (2) Credible interval of posterior distribution. (3) Risk of between-patient transfer: other patients already colonized by a pOXA-48-carrying enterobacteria each day, divided by ward. (4) Risk of within-patient transfer: patient previously colonized by a pOXA-48-carrying enterobacteria of the different species.

Supplementary Table 4

Genome assembly statistics.

Peer Review File

Source data

Source Data Fig. 5

Unprocessed number of colony-forming units (c.f.u.) obtained in the conjugation assays. The table presents the number of donor, recipient and transconjugant c.f.u. at the initial and final time. These data were used to calculate pOXA-48-like plasmids conjugation rates and frequencies.

Source Data Extended Data Fig. 8

Unprocessed number of colony-forming units (c.f.u.) obtained in the conjugation assays. The table presents the number of donor, recipient and transconjugant c.f.u. at the initial and final time. These data were used to calculate pOXA-48-like plasmids conjugation rates and frequencies.

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León-Sampedro, R., DelaFuente, J., Díaz-Agero, C. et al. Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients. Nat Microbiol (2021). https://doi.org/10.1038/s41564-021-00879-y

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