First Occurrence of the OXA-198 Carbapenemase in Enterobacterales

INTRODUCTION

Citrobacter species are Gram-negative facultative aerobic bacteria belonging to the order of Enterobacterales and family of Enterobacteriaceae (1). These species are involved in a wide variety of nosocomial infections, such as urinary tract infections, bacteremia, neonatal meningitis, and brain abscesses (2). In Citrobacter freundii infection, resistance to carbapenems is the result of (i) the overexpression of its natural cephalosporinase associated with a decrease in membrane permeability or (ii) the production of a carbapenemase. C. freundii represents the fourth enterobacterial species among carbapenemase-producing Enterobacterales in France (3). According to the data collected by the French National Reference Center, the main carbapenemases identified in Citrobacter spp. are OXA-48 and VIM-type carbapenemases (3). OXA-198 belongs to Ambler’s class D and shows weak carbapenemase activity (4). Until now, the bla_OXA-198 carbapenemase gene has been identified only in Pseudomonas aeruginosa isolates (5). In this species, this gene is embedded in a class 1 integron and carried by a plasmid of the IncP11 family (5). Here, we describe the first occurrence of bla_OXA-198 in Enterobacterales.

A carbapenem-resistant Citrobacter sp. isolate was recovered from a rectal swab during screening for carbapenemase-producing Enterobacterales intestinal carriage. The patient was an elderly man hospitalized in northern France who had no history of travel abroad. Antibiotic susceptibility testing was performed using Etest (bioMérieux, La Balme-les-Grottes, France) and broth microdilution for colistin (ThermoFisher, France) and interpreted using CLSI guidelines (6). This isolate was resistant to penicillins and broad-spectrum cephalosporins, except cefepime, and revealed decreased susceptibility to carbapenems (Table 1). Mueller-Hinton agar supplemented with cloxacillin (250 mg/liter) partially restored the susceptibility to cephalosporins, indicating an overproduction of cephalosporinase. This isolate was also resistant to amikacin, netilmicin, tobramycin, ciprofloxacin, levofloxacin, rifampin, and chloramphenicol but remained susceptible to colistin (MIC, 0.5 mg/liter), fosfomycin, and trimethoprim-sulfamide.

Carbapenemase detection tests were performed using Carba NP and NG test Carba 5 (NG Biotech, Guipry, France) and detected the presence of an OXA-48-like enzyme (7, 8). Whole-genome sequencing using Illumina technology was performed as previously described to determine the exact β-lactamase content of this isolate (9). Three β-lactamases were identified, i.e., a natural AmpC exhibiting 93.4% amino acid identity with CMY-125 and the acquired class D carbapenemases OXA-48 and OXA-198. Analysis of the genetic context revealed that the bla_OXA-48 gene was bracketed by two copies of IS1999 and carried by the canonical IncL plasmid known to be responsible for the spread of this carbapenemase (10). The bla_CMY-125-like gene was carried by the chromosome and was associated with a transcriptional regulator as described in Citrobacter spp. (11).

Transfer of bla_OXA-198 was successfully performed by electrotransformation and by conjugation as previously described (12). Since bla_OXA-198 was associated with cmlA1 (see below), chloramphenicol 30 mg/liter was used as a selector. Carba NP and carbapenem inactivation method tests were performed on Escherichia coli transformants to evaluate their efficacy in detecting OXA-198 hydrolytic activity toward carbapenems but were unable to detect any carbapenemase activity. These results highlighted the weak carbapenemase activity of OXA-198.

To obtain the full sequence of the bla_OXA-198-carrying plasmid, sequencing of the transformant E. coli TOP10 cells (pCit-OXA-198) was performed using MinION. Briefly, whole-cell DNA of the transformant E. coli TOP10 (pCit-OXA-198) was extracted using the PureLink genomic DNA kit (Invitrogen, ThermoFisher, France). Isolated DNA was controlled for quality using Nanodrop and fluorometric quantification (Qubit, ThermoFisher Scientific). The DNA library was prepared using 1 μg of genomic DNA according to the 1D library preparation protocol with the 1D native barcoding expansion kit (EXP-NBD104) and ligation sequencing kit (SQK-LSK109) (Oxford Nanopore Technologies [ONT]), including the DNA repair and end-prep step with the NEBNext formalin-fixed, paraffin-embedded DNA repair and NEBNext End repair/dA tailing module (New England Biolabs, Evry, France). The sequencing libraries were purified using Agencourt AMPureXP beads (Beckman Coulter, France), and 6 μl (63 ng) of the sequencing library was loaded onto an R9.4 FLOWMIN106 flow cell and sequenced with the MinION Mk 1B sequencing device (ONT) for 48 h. Basecalling was performed using Guppy software, available to ONT customers via their community site (https://community.nanoporetech.com), on all quality-passed reads, and contigs were then extracted using the Canu (v1.6) pipeline (13). A total of 15,469 reads were obtained with a mean size of 6,744 bp and coverage of 21×. Reads were mapped against the chromosome of E. coli TOP10 and all mapped reads were discarded. The unmapped reads gave 1 contig of 183 kb in size. This contig was finally corrected using reads from Illumina’s sequencing.

The bla_OXA-198-carrying plasmid was of 183,921 bp in size (Fig. 1A). Using PlasmidFinder 2.1 (cge.cbs.dtu.dk/services/PlasmidFinder/), this plasmid was determined to belong to the IncH1 family. Indeed, the repA gene shared 97.4% nucleotide identity with repA of plasmid R27 belonging to the IncHI family plasmids (14). Despite a known replicase, this plasmid shared very weak identity with other IncH1-type plasmids. The G+C content was of 49%. Alignment with the closest plasmids, pCFR17 (GenBank accession no. CP035277) and pBWH35 (GenBank accession no. CP020508), showed that these plasmids shared <30% nucleotide identity with pCit-OXA-198 (Fig. 1A). Note that these two plasmids do not belong to IncH1. It is surprising that the two closest plasmids did not share the same replication origin as pCit-OXA-198. In addition, pCit-OXA-198 did not share any similarities with pOXA-198 from P. aeruginosa (Fig. 1A). Plasmid pCit-OXA-198 possessed a conjugal transfer operon, two partitioning systems, and several type II toxin/antitoxin systems (ccdA/B, vapB/C, and relE/B). Interestingly, it also possessed two operons involved in metabolic functions, i.e., catabolism of glucuronide-conjugated compounds and catabolism of d-threonate and d-erythronate (15). Glucuronidation is widely used by mammals and other vertebrates to metabolize a wide range of compounds, including metabolic wastes and xenobiotics (16). A complete ABC transporter locus was also identified on this plasmid. In addition to antibiotic resistance, these results may indicate a possible role of this plasmid in the lifestyle of this isolate.

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FIG 1

(A) Circular representation of pCit-OXA-198 and comparison with closest plasmids. This representation was performed using the CGview server (http://wishart.biology.ualberta.ca). The three plasmids used for comparison were pCFR17 (GenBank accession no. CP035277), pBWH35 (GenBank accession no. CP020508), and pOXA-198 (GenBank accession no. MG958650). (B) Genetic structure surrounding the bla_OXA-198 gene. Arrows, open reading frames; triangles, inverted repeats of insertion sequences; dashed lines, homology. (C) Phylogenetic analysis at the species level of Citrobacter sp. 175G8. The phylogenetic tree was constructed using CSI phylogeny for the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/CSIPhylogeny/).

The bla_OXA-198 gene was embedded in a class 1 integron as described (4, 5). However, the gene cassette array differed from those identified in P. aeruginosa (Fig. 1B). Thus, this new integron was named In1682 according to the Integrall database (http://integrall.bio.ua.pt/). Compared to the integron of pOXA-198, seven cassettes were identified in In1682. The first cassette, aacA7, an aac(6′)-I1, was identical; but a new cassette coding a new aac(3′)-I, aacC14, was inserted in the second position (Fig. 1B). This protein corresponded to a novel cassette that shared weak identity with other aac(3′)-Is (62% to 75% amino acid identity) (Fig. 1B). The closest encoded protein was identified as AAC(3′)-I in Pseudomonas stutzeri (NCBI accession no. WP_102846203). The bla_OXA-198 was the third cassette, and another novel cassette, gcu198, was identified downstream, coding for a protein (89 amino acids in size) of unknown function. Downstream of this cassette, cmlA1 (chloramphenicol resistance) and aadA1 (aminoglycoside resistance) were present, followed by the 3′ conserved segment of the integron. Promoter P_cS mediated expression of this integron because promoter P₂ was absent. The whole structure was bracketed by a Tn3-family transposase upstream and insertion sequence IS6100 downstream (Fig. 1B). Downstream of IS6100, we identified an operon coding for mercuric compound resistance followed by a partitioning system and repA. Plasmid alignment revealed no homology between pCit-OXA-198 and pOXA-198 from P. aeruginosa (Fig. 1A).

MLST analysis (https://cge.cbs.dtu.dk/services/MLST/) revealed that this Citrobacter sp. belonged to sequence type ST128. To date, no study reporting this sequence type is present in public databases. Phylogenetic genomewide analysis was performed using CSI phylogeny (https://cge.cbs.dtu.dk/services/CSIPhylogeny/), and sequences from NCBI were used as reference genomes (Fig. 1C). The current analysis revealed that this isolate does not match other C. freundii isolates at the species level. Indeed, the closest genome corresponds to Citrobacter pasteurii UMH17. Analysis of average nucleotide identity (ANI) was performed to classify this isolate and revealed that Citrobacter sp. 175G8 actually belongs to C. pasteurii (ANI, 100%) (see Fig. S1 in the supplemental material).

To the best of our knowledge, this is the first report of OXA-198 in Enterobacterales. This carbapenemase was hidden by the presence of another carbapenemase, OXA-48. Moreover, this carbapenemase gene is not targeted by common carbapenemase detection sets and might spread silently. In conclusion, whole-genome sequencing remains the most efficient way to detect this rare carbapenemase. Of note, this isolate was recovered in northern France 10 kilometers from the border with Belgium, where the first OXA-198-producing P. aeruginosa isolate was reported, suggesting a potential regional spread of this carbapenemase and potential interspecies dissemination of bla_OXA-198.