First Report of an ST410 OXA-181 and CTX-M-15 Coproducing Escherichia coli Clone in Italy: A Whole-Genome Sequence Characterization

OXA-181 is a four amino acid substitutions derivative of OXA-48 carbapenemase, showing similar hydrolytic activity conferring resistance to penicillins and carbapenems. It was first identified in Enterobacter cloacae and Klebsiella pneumoniae from several locations in India in 2007, and further research indicated that it originated from the Gram-negative waterborne bacterium Shewanella xiamenensis. ¹ Since the first identification, OXA-181-producing Enterobacteriaceae have been reported from several countries, both in the Indian subcontinent (Bangladesh and Sri Lanka) and outside (Canada, Asia, Europe, and Africa).^11,12 The genetic environment of the bla_OXA-181 gene showed mainly a plasmid localization, including, besides IncX3, also the IncN and IncT plasmids.^7,15 Here we describe the molecular features of the first OXA-181 and CTX-M-15 coproducing Escherichia coli clinical isolate in Italy, and its identification as belonging to the phylogenetic group A and ST410 lineage.

An elderly man, previously host at a long-term care facility, was admitted on January 2017 to the emergency room of ASST Fatebenefratelli Sacco hospital (Milan) for a respiratory distress. Owing to his clinical manifestations including cardiovascular decompensation, aortic stenosis, and chronic anemia, he was transferred to the cardiosurgical ward, where rectal carriage of carbapenem-resistant E. coli (CREc) was ascertained. The surveillance rectal swab, performed at admission, resulted positive for the presence of an OXA-48-like positive Escherichia coli strain (ECS1_14) by Xpert Carba-R test (GeneExpert, Cepheid). The general conditions of the patient worsened and he underwent a cardiac valve surgical replacement. Blood and valve culture resulted negative for the growth of CREc, whereas rectal swabs remained positive. Two weeks after surgery, acute respiratory and chronic renal failures occurred and the patient died.

The ECS1_14 strain showed a multidrug resistant (MDR) profile, being resistant to cephalosporins, carbapenems, fluoroquinolones, and aminoglycosides, retaining susceptibility to fosfomycin, colistin, and tigecycline by Vitek2 System, according to EUCAST guidelines. ^a The MIC values for ertapenem (ETP), meropenem (MER), and imipenem (IMP) were determined by Etest, and a carbapenem susceptibility profile coherent with the production of an OXA-48-like enzyme was detected for ECS1_14: high-resistance levels only against ETP, a reduced susceptibility for IMP, and retained susceptibility to MER (Table 1).

The ECS1_14 genomic DNA was extracted using a QIAamp DNA minikit (Qiagen) following the manufacturer's instructions, and sequenced using Illumina Miseq with a 2 by 250 paired-end run after Nextera XT library preparation. A total of 1,454,081 reads were obtained, and assembled using the SPAdes program giving 409 contigs, of which 194 were >500 bp in length (ERX2145593).

The isolate was in silico assigned to phylogenetic group A, sequence type (ST) 410 (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/), an hyperepidemic clone identified from the three parts of the “one health” approach (humans, animals, and environment), ¹³ and known to be the founder of the globally disseminated clonal complex 23. In addition, in silico multilocus ST with the Pasteur scheme (http://bigsdb.pasteur.fr/ecoli) assigned the isolate to ST692. The ECS1_14 was genotyped using DTU web tools (www.genomicepidemiology.org/): the identified serotype was O8:H9, and the fimH subtype fimH24. The same serotype was identified as peculiar of ST410 clade C E. coli strains isolated in Germany from several sources, including companion animals, farm environment, and humans. ² The complete resistance genes content included, besides the bla_OXA-181, bla_CTX-M-15 (a globally distributed extended-spectrum β-lactamase [ESβL] gene), bla_TEM-1b (a non-ESβL determinant), bla_CMY-2 (an AmpC gene), aac(6’)-Ib-cr (an aminoglycoside acetyl transferase showing low-level resistance to aminoglycoside and fluoroquinolones), qnrS1 (conferring low-level resistance against fluoroquinolones), tetB (a tetracycline resistance gene), and sul1 (a sulfonamides-resistant determinant) genes (Table 1).

Bioinformatic analyses allowed to identify two assembly contigs highly similar (covering >99% and 100% identity) to the IncX3 pOXA181_EC14828 plasmid. Thus, polymerase chain reaction assays and Sanger sequencing were performed using the pOXA181_EC14828 as template for primers designing, obtaining the complete sequence of the pOXA181_ECS1_14 plasmid (accession number pending). Interestingly, pOXA181_EC14828, first described by Liu et al. in China, ⁶ was recently reported several times across the world (Denmark, the United Kingdom, Czech Republic, Africa, Switzerland, and Germany) from different Enterobacteriaceae (E. coli, K. pneumoniae and K. variicola) and sources (human, swine, and vegetables).^{3,5,6,9,10,14} To note, the IncX3 plasmid is linked to the dissemination of several carbapenem-resistant determinants, including bla_NDM and bla_KPC types. Interestingly, another ST692(Pasteur) E. coli strain, carrying bla_OXA-181-IncX3 plasmid and with a similar resistance profile, was reported from Burkina Faso in 2016. ⁸

The sporadic spread of OXA-48-like-producing Enterobacteriaceae has already been reported in Italy, although the prevalence of these carbapenemases remained low ⁴ ; nonetheless the isolation of an MDR E. coli isolate belonging to the widely disseminated ST410 and carrying several, transferable, resistance determinants, along with its presence in the community, is of particular concern.