On May 28, 2023, an 8-year-old child with severe high-voltage electric shock burns on 64% of his body was admitted to the Center for Traumatology and Major Burns, Ben Arous (Tunis, Tunisia). Multiple complications developed, including sepsis, urinary tract infection, and wound infections, which required the administration of broad-spectrum antimicrobial drugs (cefotaxime, imipenem, gentamicin, colistin, teicoplanin, and fosfomycin). On August 25, 2023, we transferred the patient to the burn unit of Sahloul Hospital (Sousse, Tunisia), where he received nutritional support with a high-protein high-calorie diet, transfusions for anemia, and extensive wound care and surgical skin grafting procedures. On September 20, 2023, after we isolated Morganella morganii, Proteus mirabilis, methicillin-susceptible Staphylococcus aureus, and glycopeptide-resistant Enterococcus faecium from bone fragments, the patient received piperacillin/tazobactam and cotrimoxazole for 21 days. On October 12, 2023, positive blood cultures revealed an extremely drug-resistant gram-negative bacillus we identified as Pseudomonas aeruginosa by Vitek 2.0 (bioMerieux, https://www.biomerieux.com). We conducted matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonics, https://www.bruker.com) and identified the isolate as P. guariconensis with a score of 1.84.

We determined the MICs of the P. guariconensis isolate (65411) by using broth microdilution (Sensititre; ThermoFisher, https://www.thermofisher.com) and interpreted according to clinical breakpoints of the Comité de l’Antibiogramme de la Société Française de Microbiologie/European Committee on Antimicrobial Susceptibility Testing referential (version 2024; https://www.sfm-microbiologie.org/wp-content/uploads/2024/06/CASFM2024_V1.0.pdf). MIC testing revealed resistance to nearly all antimicrobial drugs tested, including ceftolozane/tazobactam (MIC >16 mg/L), imipenem and meropenem (MIC >32 mg/L), imipenem/relebactam (MIC >16 mg/L), meropenem/vaborbactam (MIC >32 mg/L), and aztrenoam/avibactam (MIC >16 mg/L), but susceptibility to ceftazidime/avibactam (MIC <0.5 mg/L), cefiderocol (MIC <1 mg/L), and colistin (MIC <0.5 mg/L). We adjusted the treatment regimen on the basis of antimicrobial drug susceptibility results and the availability of medications in Tunisia, where cefiderocol and ceftazidime/avibactam are not yet available. We administered colistin (150,000 units/kg; 1 million units 3×/d intravenously for 25 days), along with rifampicin (20 mg/kg/d; 450 mg 1×/d intravenously for 15 days), resulting in clinical stabilization and improvement.

We reported a negative result when testing P. guariconensis 65411 by using a homemade carbaNP assay (5) and a positive result for OXA-48–like enzymes by using the lateral flow immunoassay NG-Test Carba 5 assay (NG-Biotech, https://www.ngbiotech.com) (6). We conducted short-read (Illumina, https://www.illumina.com) and long-read (Oxford Nanopore, https://nanoporetech.com) whole-genome sequencing to consolidate bacterial identification (we confirmed P. guariconensis-like-3 by using Centrifuge [7]), identify resistance genes (Center for Genomic Epidemiology, https://www.genomicepidemiology.org), and determine the bla_OXA-48-like genetic environment. We generated 1 contig of 5.6 Mbp with a guanine-cytosine content of 64.1% from a hybrid assembly by using Unicycler 0.5.0 (8), which agreed with the characteristics of reference P. guariconensis isolate ASM4082203v1 (GenBank accession no. GCF_040822035.1). We submitted the genome of isolate 65411 to the National Center for Biotechnology Information Nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore; Bioproject no. PRJNA1150136).

The resistome of P. guariconensis 65411 contained acquired genes conferring resistance to β-lactams (bla_CMY-16, bla_DHA-1, bla_OXA-1, bla_OXA-10, and bla_OXA-204), aminoglycosides [aac(6')Ib-cr, ant(2'')-Ia, aph(3'')-Ib, aph(3')-Via, aph(3')-VIb, aph(6')-Id], fosfomycin (fosA), fluoroquinolones [aac(6')Ib-cr], and additional antimicrobial drugs and biocides (catB3, ARR-3, sul1/2, qacE). High-level resistance to fluoroquinolones was because of a T83I substitution in the quinolone resistance-determining regions of GyrA. The RAST annotation identified an additional chromosome-encoded class A β-lactamase gene, named bla_GUA-1, displayed 100% sequence identity with a ß-lactamase encoded in the genome of a P. guariconensis from the Czech Republic (GenBank accession no. UQM99659.1); 72% with that of P. fulva (accession no. MBF8778391.1), P. mosselii (accession no. WP_345894069.1), and P. soli (accession no. UXZ44560.1); and 57% with the extended-spectrum β-lactamase bla_LUT-1 from P. luteola (9). All conserved motifs essential for β-lactamase activity were preserved, including the serine active site motif (S₇₀XXK), the hydrolytic water-binding site (S₁₃₀DN), and the catalytic triad component (K₂₃₄TG) (10). Those findings suggest the bla_GUA-1 gene likely encodes an intrinsic class A β-lactamase (Appendix Figure). We cloned bla_GUA-1 into an expression vector. The overexpression revealed crude protein extracts with strong nitrocefin hydrolytic activity. We confirmed the presence of a β-lactamase capable of hydrolyzing first and third generation cephalosporins (Appendix reference 1) by using ß-lactamase testing (Bio-Rad Laboratories, https://www.bio-rad.com).

Figure 1

Figure 1. Comparative genomic analysis of OXA-204 carbapenemase-positive Pseudomonas guariconensis isolated from a child in Tunisia, 2023, using P. guariconensisGenBank accession no. GCF_040822035 as the reference genome. The...

Figure 2

Figure 2. Representation of nucleotide alignment between the IS26-mediated transposon in the OXA-204 carbapenemase positive Pseudomonas guariconensisisolated from a child in Tunisia, 2023 (65411), and reference plasmids characterized...

Figure 3

Figure 3. Representation of the nucleotide alignment between the Tn2016-like transposon carrying the bla_OXA-204 gene in the OXA-204 carbapenemase positive Pseudomonas guariconensisisolated from a child...

The bla_OXA-204 carbapenemase gene is mostly detected in Tunisia (11) and in France from patients with previous travel to Tunisia. The gene is mostly found in Klebsiella pneumoniae and Escherichia coli isolates (11; Appendix reference 2), but also rarely in P. mirabilis, Citrobacter freundii, Serratia marcescens (12), Enterobacter cloacae (13), and Shewanella xiamenensis (14). The bla_OXA-204 gene is described as part of a Tn2016 transposon located on IncA/C plasmids (Appendix reference 1). In P. guariconensis 65411, the comprehensive analysis of the genetic environment of the bla_OXA-204 gene revealed the integration of a 119-kb plasmid fragment into the chromosome (Figure 1). This fragment displayed multiple resistance genes, including the β-lactamases bla_DHA-1, bla_CMY-16, bla_OXA-1, and bla_OXA-204, alongside genes conferring resistance to chloramphenicol (catB3) and aminoglycosides [aph(6')-Ic, aph(6')-Id, aph(3'')-I, aph(3')-III, aph(3')-IV, aph(3')-VI, aph(3')-VII, and aac(6')-Ib-cr]. The inserted fragment was bound by 2 IS26 elements and an 8-bp target site duplication, suggesting an IS26-mediated insertion into the chromosome. The inserted plasmid shared homology (100% homology over an 88% coverage of the 119-kbp fragment) with part of a 189,866 bp IncC plasmid originating from a K. pneumoniae isolate (GenBank accession no. CP086448) coproducing OXA-10, CMY-16, and NDM-1 (15) (Figure 2). The bla_OXA-204 gene was embedded within a 3,958 bp ISEcp1-based Tn2016-like transposon (11; Appendix reference 1) bracketed by a 5 bp target site duplication (Figure 3). In the prototypical Tn2016, ISEcp1 was disrupted by an ISKpn15 (11; Appendix reference 1), whereas in P. guariconensis 65411, it was disrupted by an IS903B generating a 9 bp target site duplication in the Tn2016-like transposon. As suggested for ISKpn15 insertion, the IS903B insertion should not interfere with the strong outward oriented promoter of ISEcp1 (11; Appendix reference 1), enabling high-level expression of bla_OXA-204.

We describe an OXA-48-like carbapenemase in Pseudomonas clinical isolate, demonstrating the dissemination of bla_OXA-48-like genes beyond Enterobacterales. OXA-204–producing P. guariconensis was initially identified as P. aeruginosa by using biochemical methods, suggesting the real occurrence of P. guariconensis might be underestimated in clinical settings. The bla_OXA-204 gene was carried by an ISEcp1-based Tn2016-like element present on a 119-kb plasmid fragment inserted into the chromosome by a large IS26-mediated composite transposon. The integrated DNA carried additional resistance determinants, including 2 cephalosporinases (CMY-16 and DHA-1) and multiple associated resistance genes, resulting in an extremely drug-resistant phenotype. At first, the bla_OXA-204 gene was characterized on a 150-kb IncA/C broad host range carrying bla_CMY-4 that could be transferred to P. aeruginosa and lead to MICs for imipenem of 8 mg/L and meropenem of 32 mg/L (11). The high level carbapenem-resistance in P. guariconenesis 65411 is likely the result of the expression of OXA-204 carbapenemase together with CMY-16 and DHA-1, which are 2 cephalosporinases known to weakly hydrolyze carbapenems (Appendix reference 3).

Our findings reinforce the critical need for ongoing surveillance, advanced diagnostic tools, antimicrobial drug stewardship, and access to novel antimicrobial drugs in low- to middle-income countries to mitigate the spread of carbapenem-resistant pathogens. Carbapenemase detection should not rely exclusively on carbapenem-hydrolytic activity such as Carba NP but also on lateral flow immunoassays for confirming the presence of carbapenemases, including OXA-48–like enzymes in nonfermenting gram-negative pathogens.