pHN7A8-related multiresistance plasmids (blaCTX-M-65,fosA3andrmtB) detected in clinical isolates ofKlebsiella pneumoniaefrom Bolivia: intercontinental plasmid dissemination?

Sir, F33:A−:B− multiresistance plasmids carrying blaCTX-M-65, fosA3 and rmtB are widespread in Escherichia coli isolates of animal origin from China.1–3 pHN7A8, isolated from a dog in 2008 and completely sequenced, is a representative of these plasmids.4 To the best of our knowledge, these epidemic plasmids have not been found in samples of human origin so far. Here we characterize two pHN7A8-related plasmids from clinical isolates of Klebsiella pneumoniae from the Bolivian Chaco region. K. pneumoniae 397Kp and K. pneumoniae 477Kp were isolated from urine samples of a 75-year-old female outpatient with liver failure and a 35-year-old female inpatient with acute diarrhoea, at the Hospital Basico Villa Montes (Villa Montes, Tarija Department, Bolivia) in June and December 2013, respectively. They showed a multiresistance phenotype including extended-spectrum cephalosporins (cefotaxime MIC > 4 mg/L; ceftazidime MIC = 8 mg/L), aminoglycosides (gentamicin MIC > 4 mg/L; amikacin MIC > 16 mg/L), fluoroquinolones (ciprofloxacin MIC > 2 mg/L; levofloxacin MIC > 4 mg/L) and, in the case of 397Kp, fosfomycin (MIC > 512 mg/L) (Table S1, available as Supplementary data at JAC Online). Clonal analysis, performed by MLST (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) and PFGE, revealed that K. pneumoniae 397Kp and K. pneumoniae 477Kp belonged to ST726, an ST recently associated with bloodstream infections in Taiwan,5 and that they were possibly related.6 Plasmids from K. pneumoniae 397Kp (p397Kp) and K. pneumoniae 477Kp (p477Kp) were transferred at high frequency (7 × 10−2 transconjugants/recipient) in conjugation experiments using E. coli J53 Azir (met pro azide resistant) as a recipient, and Mueller–Hinton agar plates plus 2 mg/L cefotaxime and 100 mg/L sodium azide for selection of transconjugants.7 Complete sequencing of both plasmids was performed using a MiSeq Illumina platform (Illumina, San Diego, CA, USA). The SPAdes assembly algorithm was used to assemble raw sequence data.8 Gaps were closed by a PCR/sequencing-based strategy, and annotation was manually performed using plasmid pHN7A8 (GenBank accession no. JN232517) as a reference. Plasmids p397Kp (76 863 bp) (GenBank accession no. LN897474) and p477Kp (74 768 bp) (GenBank accession no. LN897475) showed 100% nucleotide identity, except that p477Kp lacked the fosA3 module (Figure 1). Comparative analysis of the two F33:A−:B− plasmids from Bolivia (p397Kp, LN897474; p477Kp, LN897475) and the reference F33:A−:B− epidemic plasmid pHN7A8 (JN232517) from China. Physical maps were generated using EasyFig software.10 Genes are represented by bars and are classified by function into different groups. Homologous segments are indicated by grey shading, representing 100% (dark grey) and ≥99% (light grey) sequence identity. Numbers of SNPs, and differences involving a 9 bp repeat region in traD (open square) and a 6 bp repeat region in yddA (filled square) (both absent in the Bolivian plasmids), are indicated. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Of note, they were found to be highly related to the Chinese epidemic plasmid pHN7A8, from which p397Kp differed by only nine scattered SNPs (of which two resulted in amino acid changes in traA and traB), by the absence of a 9 bp repeat in traD and by the absence of a 6 bp repeat in yddA (Figure 1). Investigation of plasmid stability in E. coli transconjugants, carried out as previously described,7 revealed that p397Kp and p477Kp were stably maintained for up to 60 generations in the absence of selective pressure. Based on the observation that the most recently identified Bolivian plasmid (p477Kp) lacked the fosA3 resistance module, experiments were carried out to evaluate the possible loss of this module. For this purpose, E. coli J53 carrying p397Kp was maintained for 60 generations in the absence of selective pressure, and the rate of fosA3 loss was determined by replica plating of at least 100 colonies onto LB agar plates plus fosfomycin (2 mg/L) and glucose-6-phosphate (25 mg/L), and confirmation of the fosA3 presence by PCR.7 No evidence of instability of the fosA3 resistance module was observed under these experimental conditions. Results from the present study add some important information on the epidemiology of F33:A−:B− multiresistance plasmids, which were previously shown to have undergone an epidemic dissemination in China among E. coli strains infecting or colonizing animals.1–3 Indeed, p397Kp and p477Kp are the first representatives of such plasmids identified in human clinical isolates and, notably, in a clinically relevant species (K. pneumoniae), in which they have been only sporadically described previously.9 Detection of pHN7A8-related plasmids in clinical isolates from Bolivia not only underscores the ability of these plasmids to spread in the clinical setting, but also highlights their potential for intercontinental dissemination. The routes of spread of such plasmids in Bolivia are still unknown and deserve further investigation, in particular to evaluate a possible epidemiological link with China and the role of animals and the food chain. The study was supported by the European Union Seventh Framework Programme (EvoTAR; HEALTH-F3-2011-2011-282004) and by grants from the Italian Ministry for Foreign Affairs (Fortalecimiento de la red de salud del Chaco Boliviano: una perspectiva comunitaria), the Ente Cassa di Risparmio di Firenze (Florence, Italy) and the Regione Toscana (Italy) (Toscana e Chaco, 25 anni di cooperazione sanitaria: un passo decisivo verso il contenimento della diffusione delle resistenze batteriche agli antibiotici). None to declare. This work was partially presented at the International Conference on the Evolution and Transfer of Antibiotic Resistance (ICETAR), Amsterdam, The Netherlands, 2015 (Poster 36). We thank Laura Cresti and Felice Valzano for their valuable support in laboratory activities.