Work

KPC-3-producing ST167 Escherichia coli from mussels bought at a retail market in Tunisia

Year: 2017

Type: article

Abstract: Sir, KPC is one of the most prevalent and significant enzymes conferring resistance to carbapenems in Gram-negative bacteria.1 The dissemination of KPC-type carbapenemases has been associated with the spread of major clones of Klebsiellapneumoniae, such as ST258 and ST11, but horizontal transfer of blaKPC-bearing plasmids has also contributed to the emergence of KPC-producing Escherichia coli in clinics. Even though NDM, VIM, IMP and OXA-48 types have been sporadically recognized from non-human sources, the very first KPC producers of animal origin were recently reported from broilers in Egypt.2 On the other hand, KPC-producing bacteria were found in rivers and hospital wastewater, suggesting that aquatic organisms may also become a secondary reservoir of KPC enzymes. Therefore, we investigated the presence of carbapenemase-producing Enterobacteriaceae in Mytilus galloprovincialis (filter-feeding Mediterranean mussel) bought at a retail market in Tunisia. In February 2015, a batch of 200 depurated mussels was analysed. Forty pools of five mussels were each suspended in sterile peptone salt solution and the overnight culture was spread on MacConkey agar plates supplemented with imipenem. One E. coli isolate (1/40, 2.5%), as identified by the API20E gallery (bioMérieux, Marcy-l’Étoile, France), was recovered and tested positive using the Carba NP test.3,E. coli #40336 was resistant to all β-lactams, presented intermediate resistance to ertapenem and meropenem (MICs of 1.5 and 3 mg/L, respectively) and was susceptible to imipenem (MIC of 2 mg/L). This isolate was also resistant to fluoroquinolones, florfenicol, sulphonamides and trimethoprim, but susceptible to tigecycline and colistin, as tested by broth microdilution (MICs of 0.38 and 0.75 mg/L, respectively). PCR and sequencing for the blaVIM, blaIMP, blaNDM-like, blaOXA-48-like, blaKPC, blaCTX-M, blaTEM, blaSHV and blaCMY genes revealed that E. coli #40336 carried a unique blaKPC-type gene, but blaKPC-2 and blaKPC-3 variants could not be distinguished after sequencing by the PCR scheme used.4 Total genomic DNA of E. coli #40336 was extracted using a DNA purification kit (Macherey Nagel, Gutenberg, France) and quantified using a Qubit fluorometer (Invitrogen, Waltham, MA, USA). A DNA sequencing library was prepared using the Nextera XT DNA sample preparation kit (Illumina, Paris, France) and reads were de novo assembled using Velvet optimizer (http://www.illumina.com/informatics/research/sequencing-data-analysis-management/basespace/basespace-apps/velvet-de-novo-assembly-589590.html). The assembled genome was subjected to gene prediction and annotation using the website of the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/), confirming the presence of blaKPC-3 together with the blaTEM-1, blaOXA-1, blaOXA-9, aph(3′)-Ia, aadA1, strA, strB, catB3, sul1, sul2, tet(A) and dfrA14 genes. Conjugative transfer of blaKPC-3 proved unsuccessful despite several attempts and transformation did not succeed either. The blaKPC-3 gene was finally identified on an ∼180 kb IncFII plasmid (F2:A−:B− subtype) by S1-PFGE and Southern blotting with adequate probes on the native strain. The genetic environment of blaKPC-3 was first characterized using primers targeting the tnpA transposase gene, the junction between ISKpn7 and blaKPC-3, and ISKpn6, and then confirmed by the genomic sequence.5 The blaKPC-3 gene was located on the Tn4401d transposon characterized by a 68 bp deletion in the polymorphic region upstream of blaKPC-3. Interestingly, the predominance of blaKPC-3 in Portugal among K. pneumoniae, E. coli and Enterobacter spp. was also associated with the spread of an IncF plasmid carrying Tn4401d.6 Molecular analyses revealed that E. coli #40336 belonged to the ST167 phylogroup A, of the ST10 complex, also reported in US hospitals to spread blaKPC genes.7 To the best of our knowledge, this is the first report of a KPC-producing Enterobacteriaceae isolated from filter-feeding molluscs, and more globally from a food product bought at a retail market, thereby posing a potential direct threat to public health. Considering the absence of carbapenem use in animals and the global epidemiology of blaKPC plasmids and KPC-producing E. coli clones, this isolate is most likely of human origin. The mussels sampled here were grown in an area receiving hospital effluents, which is consistent with other data on KPC-producing K. pneumoniae reported from wastewater in Austria and Brazil.8,9 Together with the very recent finding of KPC-2 in clinical isolates in Tunisia,10,11 our data also support the spread of KPC-3 in humans in this country. Such an MDR KPC-producing E. coli strain in M.galloprovincialis bought at a retail market suggests that other filter-feeding mollusc species may be likely to concentrate carbapenem-resistant Enterobacteriaceae from the human reservoir. The question of whether the food chain has a role in transferring MDR organisms to humans has been raised repeatedly. Here we have shown that molluscs are likely to be an intermediate step disseminating carbapenem-hydrolysing β-lactamases most probably from hospital activities, and these enzymes could further spread back to the human community through food intake or handling. We thank Thierry Naas for providing us with a Tn4401-positive KPC-producing K. pneumoniae control strain and Lotfi Ben Romdhane for technical assistance in WGS analysis. This work was supported by the French agency for food, environmental and occupational health & safety (Anses) and also partially supported by a grant from the Fondation pour la Recherche Médicale (équipe FRM 2016, DEQ20161136698) to E. D. Y. M. has a fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche Scientifique in Tunisia. None to declare.

Source: Journal of Antimicrobial Chemotherapy

DOI: 10.1093/jac/dkx124