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.

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 bla _OXA-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-L121¹. 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 (Figure 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-48², pOXA-48_K8 carries the central part of Tn1999 inverted, with a copy of IS1 inserted in IS1999, upstream of bla _OXA-48 (named Tn1999.2 ³). Moreover, pOXA-48_K8 carries an additional copy of IS1 close to the korC gene, also found in plasmid pRA35⁴. References 1. Lu, M. C. et al. Transmission and evolution of oxa-48-producing klebsiella pneumoniae st11 in a single hospital in Taiwan. J. Antimicrob. Chemother. 75, 318–326 (2020). 2. Poirel, L., Héritier, C., Tolün, V. & Nordmann, P. Emergence of Oxacillinase-Mediated Resistance to Imipenem in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48, 15–22 (2004). 3. Carrër, A. et al. Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey. Antimicrob. Agents Chemother. 52, 2950–2954 (2008). 4. Beyrouthy, R. et al. IS1R-mediated plasticity of IncL/M plasmids leads to the insertion of blaOXA-48into the Escherichia coli chromosome. Antimicrob. Agents Chemother. 58, 3785–3790 (2014).

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 25^th and 75^th 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 25^th and 75^th 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 25^th and 75^th 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 Figure 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 Figure 5, but represented as conjugation frequency instead of rate. Source data for this figure is available as supplementary information.

Figure 1. Study population, colonised patients, and pOXA-48-carrying enterobacteria.

(a) Patients hospitalised and patients colonised by pOXA-48-carrying enterobacteria per month during the R-GNOSIS study (n= 25 months). The left panel shows the distribution of hospitalised patients per ward by month as a boxplot. Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25^th and 75^th percentiles, and whiskers extend to observations within 1.5 times the interquartile range. The right panel shows the distribution of all colonised patients per ward by month as a boxplot. (b) 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 coloured segments represent the length of hospital stay (from admission to discharge). Black outlining of colour segments indicates patient co-colonisation with more than one pOXA-48-carrying species. (c) Enterobacteria isolates carrying pOXA-48 recovered from the patients during the 25 months of the study. The species of the pOXA-48-carrying isolates are colour-coded as indicated in the legend.

Figure 2. Phylogenetic analysis of pOXA-48-carrying K. pneumoniae and E. coli.

Genetic relationships among (a) K. pneumoniae (n= 103) and (b) E. coli (n= 45) isolates carrying pOXA-48 and recovered during the R-GNOSIS study. Tree construction is based on polymorphisms in the core genome (scale: single nucleotide polymorphism [SNPs]/site). The columns to the right of the tree indicate patient code, isolate sequence type (ST), and the ward where the isolate was recovered (colour code in legend). Boxes with colour shading indicate recovery of isolates of the same sequence type (ST) from multiple patients in the hospital.

Figure 3. SCOTTI reconstruction of between-patient transfer of pOXA-48-carrying enterobacteria.

The charts represent SCOTTI-attributed between-patient transfer events involving pOXA-48-carrying enterobacteria clones in the hospital, with individual panels representing the distribution of patients colonized by pOXA-48-carrying enterobacteria on the different wards. Each row represents an individual patient, and the grey segments represent the length of stay (from admission to discharge). Coloured arrows represent transmission events predicted by SCOTTI with the highest posterior probability (see Supplementary Figures 1–4). Line colour indicates the clone responsible for the transmission event, and line thickness represents the probability of the SCOTTI-attributed transmission, as indicated in the legend: Kpn, K. pneumonia; Eco, E. coli; ST, sequence type. Numbers to the right of arrowheads indicate the number of SNPs differentiating the complete genomes of the clone pair involved in the putative transmission event. Note that the transmission events predicted by SCOTTI are not necessarily direct transmission events between patients, they can also be indirect transmission events including unobserved and non-sampled intermediate colonised patients or environmental reservoirs in the hospital.

Figure 4. Within-patient pOXA-48 transfer.

(a) Dendrogram constructed from the 21 polymorphisms present in the core region of pOXA-48. The outermost circle indicates the genus of plasmid-carrying isolates according to the colour code in the legend, the second circle indicates the isolate names, and the remaining circles indicate the presence of each plasmid SNP. Coloured boxes indicate the four pOXA-48 plasmid variants (PV1-4) carrying ‘rare’ SNPs present in clones of different species and used as genetic fingerprints. (b) Representation of patients colonized by clones carrying rare (traceable) plasmid variants. Patients are labelled with their corresponding three letter patient code. Circles represent clones isolated from the patient, with the fill colour indicating the bacterial species (see legend), and the position of the circle indicating the date of isolation. The name and sequence type (ST) of each isolate is indicated. Circles in the same row indicate different isolates of the same clone; the number inside the second circle indicates the number of SNPs accumulated in the complete bacterial genome relative to the first isolation. All isolates within each patient carried the same traceable plasmid mutation, which is indicated in the figure. Note that in patient WIX, the pOXA-48 plasmid carried the group II intron ltrA in isolate N22 but not in N11. Interestingly, in N11 ltrA is located on a different plasmid, suggesting that ltrA can easily excise/insert between genomic locations, precluding its use as a stable plasmid genetic signature (Supplementary Table 1).

Figure 5. pOXA-48 conjugation rate.

Conjugation rates of the most common pOXA-48 variant in the hospital (pOXA-48_K8) and the four core-region SNP variants used to track within-patient plasmid transfer (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 Figure 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. Source data for this figure is available as supplementary information.