Emergence of CTX-M-27-producing Escherichia coli of ST131 and clade C1-M27 in an impacted ecosystem with international maritime traffic in South America.

3 Xie M, Li R, Liu Z et al. Recombination of plasmids in a carbapenemresistant NDM-5-producing clinical Escherichia coli isolate. J Antimicrob Chemother 2018; 73: 1230–4. 4 Li R, Chen K, Chan EWC et al. Resolution of dynamic MDR structures among the plasmidome of Salmonella using MinION singlemolecule, long-read sequencing. J Antimicrob Chemother 2018; 73: 2691–5. 5 Kolmogorov M, Yuan J, Lin Y et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 2019; 37: 540–6. 6 Toleman MA, Walsh TR. ISCR elements are key players in IncA/C plasmid evolution. Antimicrob Agents Chemother 2010; 54: 3534. 7 Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006; 70: 296–316. 8 Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria. FEMS Microbiol Rev 2011; 35: 820–55. 9 He T, Wang R, Liu D et al. Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans. Nat Microbiol 2019; 4: 1450–6.

During a local surveillance study conducted to monitor the presence of WHO critical priority pathogens in impacted marine ecosystems, brown mussels (Perna perna) and oysters (Crassostrea spp.) were collected from 14 near-shore sites located at different distances from the port of Santos (the largest port of Latin America). Mussel (n " 10) and oyster (n " 10) samples, collected from each site, were placed into sterile plastic bags. The samples were kept refrigerated and processed within 3 h after collection. Following standard methods for the examination, 25 g of bivalves were distributed in sterile plastic bags containing 225 mL of Brain Heart Infusion broth and incubated at 37 C for 24 h. Subsequently, the samples were streaked onto MacConkey agar plates supplemented with ceftriaxone (2 mg/L), meropenem (2 mg/L) or colistin (2 mg/L), following incubation at 37 C for 24 h. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Two ceftriaxone-resistant E. coli isolates were recovered from mussel (E. coli 6M) and oyster (E. coli MO) samples collected from two different sites (23.987125S, 46.308609 W and 23.976040S, 46.372580 W) close to the port. Antimicrobial susceptibility testing, performed by disc diffusion and/or Etest methods, 4,5 revealed that both strains were resistant to amoxicillin/clavulanic acid, aztreonam, trimethoprim/sulfamethoxazole, ceftiofur (.32 mg/L), ceftazidime (.32 mg/L), cefotaxime (.32 mg/L) and tetracycline. Additionally, E. coli MO was resistant to nalidixic acid (.32 mg/L) and ciprofloxacin (.4 mg/L). PCR screening and Sanger sequencing revealed that these isolates were positive for the bla CTX-M-27 ESBL gene.
E. coli strains were subjected to WGS using the Illumina NextSeq (2 % 150 bp) platform (Illumina, USA). De novo assemblies were performed using Spades v. 3.11. WGS data were analysed using bioinformatics tools available from the Center for Genomic Epidemiology (www.cge.dtu.dk).
Mobilization of plasmids $130 kb in size (named pMO and p6M), bearing bla CTX-M-27 genes, was achieved by bacterial transformation using E. coli TOP10. FIB and FII replicons were identified in p6M (FAB formula F2:A#:B10), whereas FIA, FIB and FII replicon types were confirmed in pMO (FAB formula F1:A2:B20). The complete sequence of the pMO plasmid (GenBank accession no. MG886288) was obtained using de novo assembly, followed by gap closure by PCR and Sanger sequencing.
Although analysis of the genetic environment of bla CTX-M-27 genes, carried by both E. coli strains, revealed the presence of IS26 and IS903 mobile elements, E. coli MO presented a truncated IS903 upstream of the bla CTX-M-27 gene, whereas E. coli 6M presented a truncated ISEcp1 downstream of the bla CTX-M-27 gene, and tonB and ORF genes (Figure 1b).
In summary, to our knowledge, we report the first identification of CTX-M-27-producing E. coli strains, of ST131 and clade C1-M27, in Brazil. In this regard, since CTX-M-27-positive E. coli strains were recovered from areas impacted by intensive maritime traffic and transoceanic shipping activities, a possible introduction of international clones via commercial shipping routes could be speculated. 12 Another option could be polluted effluents with previously unnoticed presence of CTX-M-27-positive strains. In fact, in Brazil, aquatic environments receiving large quantities of urban wastewater, animal waste and hospital effluents have been recognized as potential sources for the dissemination of CTX-M-and carbapenemase-producing Enterobacterales. 13 Therefore, continued monitoring of ESBL-producing E. coli in South American countries remains necessary to elucidate the local epidemiology and dynamics of the transmission of high-risk clades with pandemic potential.