Unveiling the Emergence and Genetic Diversity of OXA-48-like Carbapenemase Variants in Shewanella xiamenensis

Abstract

An increase in the carbapenem-hydrolyzing capacity of class D β-lactamase has been observed in strains of multiple species, posing a significant challenge to the control of antibiotic resistance. In this study, we aimed to investigate the genetic diversity and phylogenetic characteristics of new bla_OXA-48-like variants derived from Shewanella xiamenensis. Three ertapenem-non-susceptible S. xiamenensis strains were identified, one isolated from the blood sample of an inpatient, the other two isolated from the aquatic environment. Phenotypic characterization confirmed that the strains were carbapenemase producers and exhibited antimicrobial resistance patterns to ertapenem, with some showing lower susceptibility to imipenem, chloramphenicol, ciprofloxacin, and tetracycline. No significant resistance to cephalosporins was observed. Sequence analysis revealed that one strain harbored bla_OXA-181 and the other two strains harbored bla_OXA-48-like genes, with open reading frame (ORF) similarities with bla_OXA-48 ranging from 98.49% to 99.62%. The two novel bla_OXA-48-like genes, named bla_OXA-1038 and bla_OXA-1039, respectively, were cloned and expressed in E. coli. The three OXA-48-like enzymes demonstrated significant hydrolysis activity against meropenem, and the classical β-lactamase inhibitor had no significant inhibitory effect. In conclusion, this study demonstrated the diversity of the bla_OXA gene and highlighted the emergence of novel OXA carbapenemases in S. xiamenensis. Further attention to S. xiamenensis and OXA carbapenemases is recommended for the effective prevention and control of antibiotic-resistant bacteria.

Keywords: OXA-48-like; S. xiamenensis; carbapenem hydrolysis activity; oxacillinases.

Figure 1

Identification of carbapenemase-producing strains via mCIM test. The control strain E. coli ATCC 25922 was carbapenemase negative, having a zone size of 23 mm > 19 mm. Three target isolates carried carbapenemase, which can break down 10 μg meropenem after incubation for 4 h ± 15 min at 35 °C.

Figure 2

Molecular phylogeny of the representative class D OXA in Shewanella spp. (A) Phylogenetic tree with 1000 bootstrap replicates generated using the neighbor-joining (NJ) method based on the nucleotides of this study (black bold) and typical bla_OXA genes reported in Shewanella spp. strains. The nucleotides corresponding to the OXA-48-like cluster are outlined by the dashed box. The bootstrap values are given at branching points, and the bar represents 0.05 substitutions per nucleotide position. GenBank accession numbers are given in parentheses. (B) Phylogenetic analysis with an NJ tree based on the amino acids of OXAs. Comparison of 16 CDS of the bla_OXA-48-like cluster mentioned in (A) to show the genotypic characteristics. The OXA mentioned in this study are in black bold. Bar represents 0.01 substitutions per amino acid position. Numbering is according to NCBI. (C) Alignment of amino acid sequences of the three OXA-type enzymes in this study with the amino acid sequences of OXA-48-like variants reported in Shewanella spp. from NCBI including OXA-48 (AY236073), OXA-54 (AY500137), OXA-252 (NG_050608), OXA-416 (KP264119), OXA-514 (KU866382), OXA-515 (KU866383), OXA-535 (KX828709), OXA-538 (KX827284), OXA-546 (KY682756), OXA-547 (KY684124), OXA-731 (MH718729), OXA-894 (MN525568), OXA-181 (this study), OXA-1038 (OK180617, this study) and OXA-1039 (OK180618, this study). The bottom line shows the consensus sequence of all bla_OXA genes. Dashes indicate identical residues among all the amino acid sequences. Amino acid motifs that are well-conserved among class D β-lactamases are indicated by black-outlined boxes, and the single gray-outlined box corresponds to the β5-β6 loop [36,37]. Differences in residues within three kinds of amino acids among all sequences are shaded in dark gray, and differences of more than three are shaded in light gray.

Figure 3

The MALDI-TOF MS spectra of 0.5 mg/mL meropenem. (A) The spectrum of PBS was included to rule out interference from matrix fluids. (B) Blank control spectrum of pure meropenem solution. (C) Negative control: spectrum of a carbapenemase-non-carrying strain. (D–F) Positive: OXA-181-harboring strain, OXA-1038-harboring strain and OXA-1039-harboring strain, respectively. Shaded area: [Meropenem_decarbox + H]⁺, decarboxylated degradation product of meropenem after carbapenemase hydrolysis (m/z 358.5); [Meropenem + H]⁺, meropenem molecule (m/z 384.5); [Meropenem + Na]⁺, meropenem sodium salt (m/z 406.5).

Figure 4

Results of the evaluation of the meropenem hydrolysis ability of the three OXA enzymes. (a) Verification of the hydrolysis ability of the three new enzymes (A: PBS, negative control; B: OXA-181; C: OXA-1038; D: OXA-1039). (b) Comparison of the hydrolysis rates of PBS, OXA-181, OXA-1038 and OXA-1039. All OXA enzymes showed a significant ability to hydrolyze meropenem. (c) Results of the in vitro enzyme inhibition test, which further confirmed the hydrolysis ability of the OXA enzymes. All OXA-enzyme-carrying cloning strains showed a significantly higher ability to hydrolyze meropenem compared to the non-OXA-carrying negative control (A = 25 mm).