Molecular genotyping reveals multiple carbapenemase genes and unique blaOXA-51-like (oxaAb) alleles among clinically isolated Acinetobacter baumannii from a Philippine tertiary hospital

Abstract

Background: Acinetobacter baumannii continued to be an important Gram-negative pathogen of concern in the clinical context. The resistance of this pathogen to carbapenems due to the production of carbapenemases is considered a global threat. Despite the efforts to track carbapenemase synthesis among A. baumannii in the Philippines, local data on its molecular features are very scarce. This study aims to characterize A. baumannii clinical isolates from a Philippine tertiary hospital through genotyping of the pathogen's carbapenemase genes.

Methods: Antibiotic susceptibility profiling, phenotypic testing of carbapenemase production, and polymerase chain reaction assays to detect the different classes of carbapenemase genes (class A bla_KPC, class B bla_NDM, bla_IMP, bla_VIM, and class D bla_OXA-23-like, bla_{OXA-24/40-like}, bla_OXA-48-like, bla_OXA-51-like, ISAba1-bla_OXA-51-like, bla_OXA-58-like) were performed in all collected A. baumannii, both carbapenem resistant and susceptible (n = 52).

Results: Results showed that the majority of the carbapenem-resistant strains phenotypically produced carbapenemases (up to 84% in carbapenem inactivation methods) and possessed the ISAba1-bla_OXA-51-like gene complex (80%). Meanwhile, both carbapenem-resistant and carbapenem-susceptible isolates possessed multi-class carbapenemase genes including bla_NDM (1.9%), bla_VIM (3.9%), bla_{OXA-24/40-like} (5.8%), bla_OXA-58-like (5.8%), bla_KPC (11.5%), and bla_OXA-23-like (94.2%), which coexist with each other in some strains (17.3%). In terms of the intrinsic bla_OXA-51-like (oxaAb) genes, 23 unique alleles were reported (bla_OXA-1058 to bla_OXA-1080), the majority of which are closely related to bla_OXA-66. Isolates possessing these alleles showed varying carbapenem resistance profiles.

Conclusions: In summary, this study highlighted the importance of molecular genotyping in the characterization of A. baumannii by revealing the carbapenemase profiles of the pathogen (which may not be captured accurately in phenotypic tests), in identifying potent carriers of transferrable carbapenemase genes (which may not be expressed straightforwardly in antimicrobial susceptibility testing), and in monitoring unique pathogen epidemiology in the local clinical setting.

Keywords: Acinetobacter baumannii; oxaAb; Carbapenem resistance; Carbapenemase; Philippines; β-lactamase.

Fig. 1

Summary of A. baumannii isolate characteristics based on their antibiotic susceptibility profiles in Vitek^® 2 Compact with Advanced Expert System (antibiotics used: CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; FEP, cefepime; GEN, gentamicin; IPM, imipenem; MEM, meropenem; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin–tazobactam), carbapenemase production (MHT, modified Hodge test; mCIM, modified carbapenem inactivation method; CIM-Tris, Tris-modified carbapenem inactivation method), type of bla_OXA-51-like alleles detected, presence of ISAba1 upstream the bla_OXA-51-like genes, and other carbapenemase genes detected through PCR (KPC- Klebsiella pneumoniae carbapenemase, NDM- New Delhi Metallo-β-lactamase, VIM- Verona Integron-associated Metallo-β-lactamase, OXA- Oxacillinase)

Fig. 2

Unrooted maximum likelihood tree based on the 825 nucleotides of the bla_OXA-51-like genes of the study isolates and known references (Additional File 1) using the three-parameter (TPM1—unequal frequencies) with gamma distribution model of DNA substitution. Only the general tree topology was shown. Genes were clustered based on the subclades shown in the bla_OXA-51-like gene tree generated in the Beta-Lactamase Database-Structure and Function (http://www.bldb.eu/; accessed on 13 March 2023). Samples marked with red circles are alleles matching with known bla_OXA-51-like variants, while those with black triangles are the unique alleles. Most of the rare alleles clustered under the bla_OXA-66 subclade under the international clonal complex 2 in sequence-based typing

Fig. 3

Amino acid linkage map of the unique bla_OXA-51-like alleles from the study. The amino acid substitution nomenclature indicates the original amino acid followed by the position and the replacement. The substitutions are relative to the reference bla_OXA-66 sequence. Boxes colored yellow indicate alleles with amino acid changes in a predicted active site residue (K217)