Chromosomal 16S Ribosomal RNA Methyltransferase RmtE1 in Escherichia coli Sequence Type 448.

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone.

RmtE (RmtE1 and its variant RmtE2) is an uncommon plasmid-mediated 16S rRNA methyltransferase (16S RMTase) found in gram-negative bacteria; only 4 strains have been reported to produce RmtE, all Escherichia coli, including 1 from the University of Pittsburgh Medical Center (Pittsburgh, PA, USA) (–). We report the genetic context of rmtE (rmtE1) in another E. coli clinical strain identified at this hospital.

E. coli YDC774 was identified in 2016 in the urine of a local elderly man with a history of bladder cancer for which he had undergone transurethral resection of the bladder and completed chemotherapy. He had E. coli urinary tract infection treated with ciprofloxacin 3 months earlier; further details were unavailable. E. coli YDC774 was resistant to cefotaxime, levofloxacin, ciprofloxacin, and trimethoprim/sulfamethoxazole and susceptible to ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, minocycline, and colistin. The strain was highly resistant to amikacin (MIC >32 μg/mL), gentamycin (MIC >16 μg/mL), and tobramycin (MIC >8 μg/mL). Because the positive culture was believed to represent asymptomatic bacteriuria, the patient was not treated with antimicrobial drugs.

We aimed to elucidate the genetic context of rmtE in E. coli YDC774. Although rmtE has been identified exclusively on plasmids, neither broth conjugation with E. coli J53 nor transformation of E. coli TOP10 with purified plasmids mobilized rmtE, leading us to speculate the gene might be located on the chromosome. We therefore sequenced the YDC774 genome with PacBio RS II sequencing instrument (Pacific Biosciences, Menlo Park, CA, USA) as described (). Sequencing with a single SMRT cell yielded 64,878 reads averaging 10,991 bp. De novo assembly generated 8 contigs; the largest was ≈4.3 Mbp, which had ≈122× coverage and was consistent with a large portion of the E. coli chromosome.

E. coli YDC774 belonged to sequence type (ST) 448 by in silico multilocus sequence typing. E. coli ST448 has been reported in recent years among extended-spectrum β-lactamase– and New Delhi–type metallo-β-lactamase–producing strains (,). The chromosomal contig contained rmtE (rmtE1 allele), blaCMY-2, aac()-VIa, aadA, strA/B, floR, sul1, sul2, tet(A), and dfrA7 as determined by ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). We identified no resistance genes on the other contigs, including those representing a 96-kb IncY plasmid resembling p12579_1 (GenBank accession no. CP003110.1) in enteropathogenic E. coli strain RM12579 (99% identity over 83% coverage). Several other 16S RMTase genes, such as rmtB, rmtC, and rmtF, have been found on the chromosome of gram-negative bacteria (,).

The region surrounding rmtE1 was annotated with Rapid Annotations by using Subsystem Technology server (http://rast.nmpdr.org) and curated manually by using blastn and blastp (http://blast.ncbi.nlm.nih.gov/blast) to elucidate the context of its chromosomal integration. Using E. coli ATCC 25922 as the reference genome, we determined that a 101,257-bp sequence was inserted in an intergenic region between the 4′-phosphopantetheinyl transferase gene and the NAD(P)H nitroreductase gene on the E. coli chromosome.

This inserted sequence can be divided into 2 regions. The first comprises several inserted sequences, such as IS186, ISCR1, and 1 antimicrobial resistance gene, aadA. Downstream of this first region, the inserted fragment is similar to that in pYDC637, an IncA/C plasmid carrying rmtE1 also found at the University of Pittsburgh Medical Center in 2012 (Technical Appendix Figure) (). However, the second region comprises 3 small fragments. The first contains aadA1-bx, 4 mobile elements, and several other genes and is in reverse orientation from that of pYDC637. The second small fragment harboring blaCMY-2 is identical to that found in the core region in pYDC637 (Technical Appendix Figure) and also is in reverse orientation from the corresponding region of pYDC637. The third small fragment harboring rmtE is located in the acquired region of pYDC637. This finding suggests that, on mobilization into the chromosome, gene rearrangements occurred among these fragments. The region between 2 hypothetical proteins appears to have been deleted at or after integration, which includes genes involved in plasmid replication and conjugative transfer (Technical Appendix Figure).

rmtE1 is bound by an ISCR20-like element and an IS1294-like insertion sequence. This immediate unit is identical to that found in pYDC637. ISCR20 and IS1294 belong to IS91 family, which is considered related to some antimicrobial drug resistance genes, including 16S RMTase genes, which appears to have been the case in the mobilization of rmtE1 as well. We could not identify direct repeats upstream and downstream of the unit that would define the exact boundary of this unit. In comparing the genetic context of rmtE1 and rmtE2, ISCR20-like transposase is located upstream of rmtE1 and rmtE2 (GenBank accession nos. KT428293 and NZ_LRIX01000127). However, the transposase genes located downstream of the 2 16S RMTase genes are distinct. The genetic environment of rmtE2 is identical between the 2 plasmids from China (GenBank accession no. KT428293) and Canada (GenBank accession no. NZ_LRIX01000127).

In summary, we identified chromosomal integration of rmtE1, an unusual 16S RMTase, and blaCMY-2, a commonly observed acquired AmpC β-lactamase, in an E. coli ST448 clinical strain, an event that generated stable co-resistance to aminoglycosides and oxyiminocephalosporins. We found no evidence of further spread of this strain in the hospital. Nonetheless, the findings underscore E. coli ST448 as a potential high-risk multidrug-resistant E. coli clone.

Technical Appendix

Comparative analysis of the rmtE1-carrying region of the chromosome of Escherichia coli YDC774 and pYDC637 from E. coli YDC637.