First Report of OXA-181-Producing Enterobacterales Isolates in Latin America

ABSTRACT We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the β-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

From June 2019 to September 2021, five carbapenem-resistant enterobacterial isolates were obtained from different patients at two health care institutions in Lima, Peru. KP1137, KP1139, and EC1141 were isolated from urine; KP1138 and CP1140 were isolated from blood and bronchial secretions, respectively. Identification and susceptibility testing were performed on the Vitek2 compact system (bioMérieux, France). The results were analyzed using criteria from the Clinical and Laboratory Standards Institute (CLSI) (14). Phenotypic detection of carbapenemases was performed using the Triton-Hodge test (15). Detection of OXA-48-like carbapenemase production was performed using the RESIST-4 OKNV (OXA-48-like, KPC, NDM, VIM) immunochromatographic lateral flow assay (Coris BioConcept, Belgium) following the manufacturer's instructions. Molecular confirmation of OXA-48-like genes was performed by conventional PCR using the primers 59-ATGCGTGTATTAGCCTTATCGG-39 (forward) and 59-TGAGCACTTCTTTTGTGATG-39 (reverse), yielding a 775-bp amplicon (16).
The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). K. pneumoniae isolates KP1137 and KP1138 were resistant to ertapenem and susceptible to second-, third-, and fourth-generation cephalosporins. KP1139 was resistant to all b-lactams, including ertapenem, imipenem, and meropenem. It was also resistant to fluoroquinolones, tetracyclines, and sulfamethoxazole/trimethoprim, only showing susceptibility to aminoglycosides. The isolates E. coli EC1141 and C. portucalensis CP1140 were resistant to ertapenem (see Table S1 in the supplemental material).
All isolates were identified as carbapenemase producers using the Triton-Hodge test. Moreover, all were positive for the RESIST-4 OKNV immunochromatography test and bla OXA-48 -like genes using conventional PCR. All isolates had multidrug-resistant (MDR) phenotypes. MDR bla OXA producers are a rising health care issue that lengthens hospital stays and comorbidities because of their resistance to the antibiotics used to treat CPE infections (24).
The bla OXA-181 gene has been reported in K. pneumoniae (13), E. coli (25), and Citrobacter sp. (26) isolates. The plasmid incompatibility group IncX3 was identified in all our isolates, in line with previous studies in CPE (27). This plasmid group is involved in the worldwide dissemination of bla OXA-181 (28), and its presence in these CPE isolates suggests extended dissemination of bla OXA-181 in Peru. The bla OXA-181 gene was located in a truncated transposon Tn6361, flanked by two DIS26 insertion sequences. The qnrS1 gene was detected downstream of bla OXA-181 , conferring fluoroquinolone resistance in all isolates. The bla OXA-181 gene was flanked upstream by the Tn3-like DIS3000 truncated insertion sequence, followed by the truncated ISEcp1 gene, and downstream by the DlysR (transcriptional regulator)-Dere (erythromycin esterase)-DrepA (Col replicase) gene cluster (Fig. 2). This fragment is followed by the ISKpn19-tnpR-qnrS1-IS2-like gene set, whose structure is similar to Tn6292. This region was also compared to pRIVM_C011701_1 (GenBank accession number CP068340; 100% coverage and 100% sequence similarity) and pKP709-OXA181 (MN227183; 100% coverage and 100% sequence similarity).
In summary, we present the first report of MDR OXA-181-producing Enterobacterales isolates from Peru and highlight the use of WGS to monitor the dissemination of CPE isolates. Our results suggest the emergence and wide distribution of bla OXA-181 in association with the qnrS1 gene on IncX3-type plasmids, which could represent the primary vector for the spread of bla OXA-181 in Latin America. Ethical approval. The study protocol was approved by the Institutional Ethics Committee of the Universidad Peruana Cayetano Heredia (SIDISI 207858). Patient information was anonymized and deidentified before analysis.
Data availability. The raw read files and assemblies for the five isolates are available at NCBI under BioProject accession number PRJNA860216.

ACKNOWLEDGMENTS
The study was funded by Prociencia grant number 088-2018. D.C., G.S., and P.T. are supported by a D43 TW007393 training grant awarded to UPCH by the Fogarty International Center of the U.S. National Institutes of Health. We thank Alejandra Dávila-Barclay for her invaluable input and support with data visualization.