Whole-Genome Characterization of a Shewanella algae Strain Coharboring bla_CTX-M-15 and armA Genes on a Novel IncC Plasmid

LETTER

Multidrug-resistant Shewanella species strains are emerging worldwide (1, 2). Here, we describe an unusual Shewanella algae (18064-CSB-B-B) isolated from chicken stool collected in July 2018 on the island of Zanzibar (Tanzania) during an ongoing study (http://p3.snf.ch/Project-170063).

The sample was screened by implementing selective enrichments (3). Species identification was confirmed by analyzing the whole-genome sequence (WGS) with the tools of the Type (Strain) Genome Server (https://tygs.dsmz.de/) (4, 5). The following MICs were obtained by implementing the Sensititre GNX2F/ESB1F panels (Thermo Fisher): cefoxitin, ≤4 μg/ml; ceftazidime, 4 μg/ml; cefotaxime, 64 μg/ml; ceftazidime-clavulanate and cefotaxime-clavulanate, ≤0.12 μg/ml; cefepime, ≤2 μg/ml; meropenem, ≤0.5 μg/ml; ciprofloxacin, ≤0.25 μg/ml; gentamicin, >16 μg/ml; amikacin, >32 μg/ml; trimethoprim-sulfamethoxazole, >4/76 μg/ml; doxycycline, ≤2 μg/ml; and colistin, ≤0.25 μg/ml.

WGS was achieved by combining MinION (Oxford Nanopore) and NovaSeq-6000 (Illumina) as previously described (6). The output was interpreted by implementing the tools of the Center for Genomic Epidemiology (www.genomicepidemiology.org/) and PubMLST (www.pubmlst.org/plasmid/). A 149,553-bp IncC (formerly A/C₂) type 2 ST1 plasmid (p18064-CSB-B-B) coharboring bla_CTX-M-15, armA, sul1, and dfrA12 antibiotic resistance genes (ARGs), was identified (7). The bla_CTX-M-15 was flanked upstream by insertion sequence ISEcp1, whereas armA was 56,750 bp apart from bla_CTX-M-15 and within the ARI-A antibiotic resistance island (see Fig. S1 and S2 in the supplemental material) (8). In particular, armA was flanked upstream by ISCR1 followed by a partial ISEc28 and downstream by two hypothetical proteins trailed by ISEc29 and IS26. Moreover, a class I integron carrying dfrA12 was located downstream of ISCR1 and separated by sul1. Overall, the integron and armA regions were flanked upstream by IS5075 and Tn1696 and downstream by Tn2-IS4321 elements. The transposon appeared nonfunctional, because no target site duplications were identified (Fig. 1).

FIG 1.

Linear map of the armA resistance region of p18064-CSB-B-B found in S. algae (image generated with SnapGene Viewer 5.0.4). Comparison with similar IncC plasmids identified by BLASTn analysis in Providencia rettgeri and E. coli and in the chromosome of several A. baumannii strains (strain 6507, isolated in India and sequenced in 2018, was selected as the prototypic genome among others found with BLASTn analysis). Arrows, open reading frames: red, resistance genes; yellow, transposase genes; turquoise, class 1 integrase; white, other genes.

BLASTn analysis showed that the ISCR1-ISEc28-armA-ISEc29 element was previously reported in 19 IncC plasmids from Enterobacterales and in 107 Acinetobacter baumannii isolates, of which 8 were on plasmids negative for Enterobacterales replicons (data not shown).

Several IncC plasmids showed a high level of identity with p18064-CSB-B-B, although none of them possessed both the bla_CTX-M-15 and armA regions (see Fig. S1). p18064-CSB-B-B was compared with 263 deposited IncC plasmids with 100% nucleotide identity to the replicase gene of reference plasmids pR148 and pR55 (8). The phylogeny tree indicated that p18064-CSB-B-B belonged to a node containing 4 plasmids (see Fig. S3 in the supplemental material). The p18064-CSB-B-B backbone was related to that of the NDM-1 plasmid (GenBank accession no. LN831185) identified in Vibrio cholerae in New Delhi (9). The other 2 IncC plasmids were from Escherichia coli strains isolated in the United States (GenBank accession no. CP029118) and Canada (GenBank accession no. CP012902) (10).

The circular 4,744,471-bp chromosome of 18064-CSB-B-B contained the carbapenemase bla_OXA-55 in a genetic environment already reported in two S. algae isolates (see Fig. S4 in the supplemental material). In this context, we note that Shewanella species isolates are suggested to be the natural progenitors of several bla_OXA genes (2, 11–13). The same accounts for qnrA3, which was the only additional ARG present on the chromosome of 18064-CSB-B-B (14).

We described the first IncC plasmid coharboring bla_CTX-M-15 extended-spectrum β-lactamase and armA 16S rRNA methylase ARGs. Thanks to its great ability to be mobilized, the bla_CTX-M-15 ISEcp1 was already observed in several IncC plasmids (7, 15). On the other hand, the aminoglycoside resistance gene armA was located in a genetic context that is rare on IncC plasmids but frequent in A. baumannii isolates.

IncC plasmids have not been described in Acinetobacter species isolates, and we did not identify complete IncC plasmids in GenBank. However, the region comprised between 1 to 24,748 and 135,839 to 149,553 nucleotide positions of p18064-CSB-B-B, encoding the transfer locus, hypothetical proteins, restriction methylase, and partitioning protein ParB but not the replicase gene, is present in the chromosome of A. baumannii PB364 (GenBank accession no. CP040425). This is important and novel information suggesting that IncC plasmids can enter Acinetobacter spp. but are probably not stable.

Accession number(s).

Plasmid p18064-CSB-B-B and the chromosome of S. algae strain 18064-CSB-B-B were deposited under GenBank accession no. CP047421 and CP047422, respectively.