The whole-genome sequencing analysis revealed a polyclonal dissemination of NDM-1 and NDM-9 variants in Escherichia coli (n = 20) and Klebsiella pneumoniae (n = 2) in Tahiti since 2015 via interspecies transfer of three different blaNDM-carrying plasmids (IncR, IncHI2, and IncF) and patient-to-patient cross-transmission. It highlights the potential risk of importation of NDM producers in France, where French Polynesia is not considered stricto sensu a foreign country from which repatriated patients have to be screened.
KEYWORDS: NDM-1, NDM-9, epidemiology, Escherichia coli, Tahiti, NDM-1, whole-genome sequencing
ABSTRACT
The whole-genome sequencing analysis revealed a polyclonal dissemination of NDM-1 and NDM-9 variants in Escherichia coli (n = 20) and Klebsiella pneumoniae (n = 2) in Tahiti since 2015 via interspecies transfer of three different blaNDM-carrying plasmids (IncR, IncHI2, and IncF) and patient-to-patient cross-transmission. It highlights the potential risk of importation of NDM producers in France, where French Polynesia is not considered stricto sensu a foreign country from which repatriated patients have to be screened.
INTRODUCTION
NDM-1 (New Delhi metallo-β-lactamases) was first reported in Sweden in 2008, produced by a Klebsiella pneumoniae isolate recovered from a patient repatriated from India (1). Since then, NDM-producing Enterobacterales isolates have disseminated worldwide, representing a serious challenge for treatment, infection control, and public health. In Enterobacterales, the current spread of the blaNDM-1 gene was not related to the spread of specific clones. Indeed, this gene has been identified in a large variety of enterobacterial species (e.g., Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., and Salmonella) carried on many different plasmids (IncF, IncA/C, IncL/M, IncH, IncN, and IncX3) (2). To date, 29 variants of NDM have been reported (3), among which NDM-1 is the most prevalent (4–6). However, NDM-5 was found to be increasingly prevalent in E. coli isolates in several countries, including India (7), Myanmar (8), China (9), and Italy (10). Overall, NDM-producing Enterobacterales infections are considered endemic to the Indian subcontinent. In addition, many sporadic cases have been described in China and Australia (11). However, the epidemiology of the NDM producer in French Polynesia, a hot spot for tourism, remains partially unknown.
In the Tahiti hospital (440 beds), 9,000 rectal swab screenings are performed each year. This screening policy, which aims to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacterales- and carbapenemase-producing Enterobacterales (CPE)-colonized patients, is performed once per week for all hospitalized patients. The average rate of ESBL is relatively low, at 5%. In addition, from 2015 to 2019, only 29 carbapenemase-producing Gram-negative isolates, including the 22 NDM-producing Enterobacterales and 7 carbapenemase-producing Acinetobacter baumannii (4 OXA-23 and 3 NDM-1) cases (12), have been detected from screening and clinical samples. The 22 NDM-producing Enterobacterales isolates (20 E. coli and 2 K. pneumoniae) have been sent to the Associated French National Reference Center (NRC) of Antimicrobial Resistance for expertise on resistance mechanism. These NDM producers were isolated from rectal swab (n = 16), urine (n = 2), wound (n = 1), peritoneal fluid (n = 1), and hospital environment (n = 1) samples (Table 1). All of these NDM-producing isolates harbored a positive Carba NP test (13) and a positive NDM signal using the NG-test Carba 5 (NG Biotech, Guipry, France) (14).
TABLE 1.
Characteristics of NDM-producing E. coli and K. pneumoniae
| Characteristica |
E. coli isolate name |
K. pneumoniae isolate name |
||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 81E2 | 84C4 | 889H1 | 91I3 | 94C4 | 98I2 | 103C9 | 104C8 | 116J2 | 130B8 | 130H5 | 131D10 | 134F3 | 136E10 | 136J2 | 141J8 | 144C7 | 144D2 | 146F6 | 148B3 | 51B4 | 156B3 | |
| Sample | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Urine | Rectal swab | Peritoneal fluid | Wound | Rectal swab | Rectal swab | Rectal swab | Rectal swab | Potty chair | Rectal swab | Rectal swab | Urine | Urine |
| Isolation date | May 2015 | July 2015 | Sept. 2015 | Sept. 2015 | Oct. 2015 | Dec. 2015 | Feb. 2016 | Feb. 2016 | Aug. 2016 | Jan. 2017 | Jan. 2017 | Jan. 2017 | March 2017 | March 2017 | March 2017 | June 2017 | June 2017 | July 2017 | July 2017 | Aug. 2017 | May 2014 | Nov. 2017 |
| Patient characteristic | ||||||||||||||||||||||
| Age (yr) | 50 | 20 | 70 | 47 | 67 | 54 | 44 | 57 | 48 | 79 | 66 | 73 | 69 | 82 | 62 | 72 | 86 | - | 47 | 69 | 56 | 60 |
| Sex | F | M | F | M | F | M | M | F | M | M | M | M | F | F | M | M | F | - | M | M | M | F |
| Hospital unit | Visceral surgery | Hematology | Nephrology | Hematology | Pneumology | Visceral surgery | Visceral surgery | Cardiology | Internal medicine | outpatient | Neurology | Outpatient | Intensive care unit | Pneumology | Hepatogastroenterology | Internal medicine | Cardiology | Internal medicine | Internal medicine | Internal medicine | Internal medicine | Outpatient |
| Carba NP test | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Carbapenemase | NDM-1 | NDM-1 | NDM-1 | NDM-1 | NDM-1 | NDM-1 | NDM-9 | NDM-1 | NDM-1 | NDM-1 | NDM-1 | NDM-9 | NDM-1 | NDM-9 | NDM-9 | NDM-1 | NDM-9 | NDM-1 | NDM-9 | NDM-9 | NDM-1 | NDM-9 |
| ESBL | CTX-M-3 | CTX-M-3 | CTX-M-14 | CTX-M-3 | CTX-M-14 | CTX-M-14 | CTX-M-15 | CTX-M-15 | ||||||||||||||
| Antimicrobial susceptibility testing, by drugb | ||||||||||||||||||||||
| Amoxicillin | R | R | R | R | R | R | R | R | R | R | R | S | R | R | R | R | R | R | R | R | R | R |
| Amoxicillin + CLA | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ticarcillin | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ticarcillin + CLA | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Piperacillin | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Piperacillin + TZB | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Cefotaxime | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ceftazidime | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ceftazidime + AVI | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Cefepime | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ceftolozane + TZB | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Aztreonam | S | S | R | R | S | R | S | R | R | S | S | S | S | S | S | S | S | S | R | S | R | R |
| Meropenem | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Imipenem | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Ertapenem | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Gentamicin | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Amikacin | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | R | S |
| Tobramycin | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R |
| Colistin | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
| SXT | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | R | S | R |
| Levofloxacin | R | S | S | R | S | R | R | S | R | R | R | R | R | R | R | R | S | R | S | S | S | R |
| Ciprofloxacin | R | S | S | R | S | R | R | S | R | R | R | R | R | R | R | R | S | R | S | S | S | R |
| Fosfomycin | R | S | S | R | R | S | R | S | S | R | R | R | R | R | R | R | R | R | R | R | R | R |
CLA, clavulanate; TZB, tazobactam; AVI, avibactam; SXT, trimethoprim-sulfamethoxazole.
R, resistant; S, susceptible.
Antimicrobial susceptibility testing of the 22 NDM-producing isolates was performed by disk diffusion, and MICs of colistin were determined by broth microdilution. Results were interpreted according to EUCAST guidelines updated in 2020 (http://www.eucast.org). Results are recapitulated in Table 1. All isolates were resistant to all β-lactams, including carbapenems, except for aztreonam for 14 E. coli isolates that do not express any associated ESBL (Table 1). All NDM-producing isolates remained susceptible to colistin. Amikacin also remained a therapeutic option for most (21/22) of the isolates.
Whole-genome sequencing (WGS) was performed and analyzed as previously described (15). WGS analysis revealed polyclonal dissemination of NDM-producing E. coli (Fig. 1) and K. pneumoniae. Indeed, the two K. pneumoniae isolates were unrelated, since they belonged to two different sequence types (STs), i.e., ST22 and ST307. In regard to NDM-producing E. coli isolates, we identified eight different STs (ST10, ST48, ST167, ST190, ST206, ST226, ST744, and ST5204). Of note, all NDM-1-producing E. coli isolates of ST167 corresponded to the dissemination of a single clone (<90 single-nucleotide polymorphisms between isolates) (Fig. 1; see also Fig. S1 in the supplemental material). In contrast, ST206, ST226, and ST744 included two, two, and three clones, respectively (Fig. 1 and Fig. S1).
FIG 1.
Phylogenetic analysis of NDM-producing E. coli isolates, French Polynesia, 2015 to 2017. The scale bar (0.07) corresponds to the number of single-nucleotide polymorphisms per position of common sequences.
Mating-out assays were performed for all isolates as previously described using agar plates supplemented with cefoxitin (10 μg/ml) and sodium azide (100 μg/ml) for the selection of transconjugants (16). Plasmid typing was determined from WGS data of transconjugants using the PlasmidFinder tool available on the Center for Genomic Epidemiology server. Transconjugants were obtained for all clinical isolates. WGS of transconjugants and Kieser (17) plasmid extraction revealed that the blaNDM-1 gene is carried on an ∼50-kb plasmid of IncR type, whereas the blaNDM-9 gene is carried on an ∼160-kb plasmid of IncHI2 type or an ∼130-kb plasmid of IncF type. These three of plasmids are not specific to a single ST, demonstrating in vivo transfer of these plasmids. Accordingly, the concomitant outbreaks of NDM-1- and NDM-9-producing E. coli relied on a mix of plasmid transfer and bacteria cross-transmission from patient to patient. Regarding the two NDM-producing K. pneumoniae isolates, blaNDM-1 and blaNDM-9 are both carried on an IncF-type plasmid.
Our study highlighted the cooccurrence of NDM-1 and NDM-9 in Tahiti. Contrary to NDM-1, which is disseminated worldwide, the NDM-9 variant was reported only a few times in Asia (China, South Korea, Taiwan, India) (9, 18–20) and one time in Switzerland in K. pneumoniae isolated from wastewater (21). Of note, epidemiological investigations revealed that no patients colonized with NDM producers have travelled outside of French Polynesia, suggesting possible dissemination in the community or contamination through a hospital environmental source, since patients are transferred weekly between the Tahiti and Raiatea hospitals (250 km away). In addition, such polyclonality has already been observed in Tahiti for carbapenemase-producing Acinetobacter spp. recovered from environmental sources in the Tahiti hospital (12). However, implementation of a cohorting unit in the Tahiti hospital associated with the systematic screening of patients and specific decontamination of the potential environmental sources of dissemination (mattress, siphon) around each CPE-colonized patient might be powerful in limiting the spread of NDM-producing Enterobacterales in French Polynesia, as well as in countries where patients are regularly repatriated (mainly Metropolitan France).
Here, we highlight the potential (low but existing) risk of importation of NDM-1 and NDM-9 producers from French Polynesia. This risk is of particular importance in France, since French Polynesia is not stricto sensu considered as a foreign country from which repatriated patients have to be screened for CPE colonization.
Data availability.
Sequencing reads from the 22 sequenced NDM-producing isolates have been deposited in the GenBank database under BioProject number PRJNA660947.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the Assistance Publique – Hôpitaux de Paris and the Université Paris-Saclay.
Footnotes
Supplemental material is available online only.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Sequencing reads from the 22 sequenced NDM-producing isolates have been deposited in the GenBank database under BioProject number PRJNA660947.