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Department of Microbiology and Infectious Diseases, Concord Hospital, NSW Health Pathology, Sydney, NSW, AustraliaSchool of Medical Sciences, Department of Infection and Immunity, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
Department of Microbiology and Infectious Diseases, Concord Hospital, NSW Health Pathology, Sydney, NSW, AustraliaConcord Hospital Clinical School, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
We report the isolation of a multidrug resistant Acinetobacter bereziniae isolate harbouring a plasmid blaNDM-1 in central Sydney, Australia from a patient who had travelled to the Philippines. While carbapenem-resistant Acinetobacter baumannii complex (e.g., A. baumannii, A. calcoaceticus, A. baumannii, A. dijkshoorniae, A. nosocomialis, A. pittii and A. seifertii) (CRAB) group have been previously described, carrying the blaNDM-1,
Australian Commission on Safety and Quality in Health Care National Alert System for Critical Antimicrobial Resistances (CARAlert): Laboratory Handbook.
Acinetobacter berezinae is an environmental Acinetobacter species of the non-baumanii complex, with some routine medical biochemical databases such as the Vitek 2 XL unable to identify it correctly. Conversely, matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) offers a rapid and convenient identification method for routine laboratories,
Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS system for the identification of Acinetobacter species from blood cultures: comparison with VITEK 2 and MicroScan systems.
and has been shown here to accurately identify an A. bereziniae isolate as confirmed by molecular methods. Furthermore, A. bereziniae harbouring a plasmid blaNDM-1 described here highlights the potential role of non-baumannii Acinetobacter disseminating antibiotic resistance genes through hospital environments.
The Acinetobacter bereziniae isolate carrying the metallo-β-lactamase resistance gene was isolated from the rectal swabs collected during infection control screening after the patient's recent hospitalisation in the Philippines. The organism did not grow on ESBL Brilliance agar (ThermoFisher, Australia) but grew on CRE Brilliance agar (ThermoFisher) as clear, slightly pigmented yellow colonies incubated 24–48 h at room temperature as instructed by the manufacturer (Fig. 1). The organism growing on the selective CRE Brilliance agar media was a Gram-negative coccobacillus by Gram stain and sub-cultured on horse blood agar (ThermoFisher) for further identification and susceptibility testing. The organism was first identified as Acinetobacter bereziniae by MALDI-TOF MS (Bruker, Australia) with a score of 2.53. This was a good reliable result using this identification system since a score value ≥2 indicated species identification; a score value between 1.7 and 1.9 indicated genus identification, and a score value <1.7 indicated no identification. Identification using the Vitek 2 XL (bioMerieux, Australia) system was also performed by using the GNI ID card with reference code 21341 (bioMerieux). After 9.92 hours, the automated identification system gave an excellent identification as Acinetobacter lwoffii with a probability of 99% with a Bionumber of 0001011101500102. The identification as Acinetobacter bereziniae was later confirmed using 16S rRNA sequencing. Results showed a 100% match using sequence data assembled and compared with previously reported sequence by using the basic local alignment search tool (BLAST) of the National Centre for Biotechnology Information (NCBI) database.
Fig. 1Acinetobacter bereziniae carrying the blaNDM-1 plasmid gene growing on CRE Brilliance agar (Thermofisher) as clear, slightly pigmented yellow colonies incubated 24–48 h at room temperature.
On further testing the RAPIDEC CARBA NP (bioMerieux) was positive for the isolate which was indicative of the presence of a carbapenemase gene. Molecular testing using the gene Xpert Carba-R (Cepheid, Australia), a real-time polymerase chain reaction assay for rapid detection and differentiation of five genes (blaKPC, blaVIM, blaOXA-48, blaIMP-1, blaNDM) revealed the presence of a blaNDM carbapenemase gene. The isolate was later referred to a reference laboratory, the Institute of Clinical Pathology and Medical Research (ICPMR) in Westmead Hospital, Sydney, for whole genome sequencing which confirmed the presence of blaNDM-1 and further classified it as ST1318. Carbapenem resistance and further susceptibility of the organism to other antibiotics were confirmed using Vitek 2 XL, Etest strips (bioMerieux) and disc-diffusion (Thermofisher) susceptibility testing using both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) guidelines.
Colistin susceptibility was confirmed by broth microdilution (BMD) using the MICRONAUT MIC-Strip (MERLIN Diagnostika, Dutec, Australia). BMD quality control was performed with both E. coli ATCC 25922 and E. coli NCTC 13846 (mcr-1 positive). Acinetobacter bereziniae antibiotic susceptibility results are shown in Table 1.
Table 1Antibiotic resistant profile of the Acinetobacter bereziniae carrying the blaNDM-1 gene
Acinetobacter species are ubiquitous in nature, and their ability to survive in many ecological niches is worrisome, especially when carrying multidrug-resistant genes in a hospital environment. Acinetobacter bereziniae, also referred to as Acinetobacter genospecies 10, has been isolated in sewage in Denmark, environmental surfaces in Korea, vegetables in Hong Kong and UK, meat and animals in Lebanon, as well as human skin in Germany and Hong Kong.
The first A. bereziniae carrying the blaNDM-1 gene was reported in Brazil in 2014 from a 69-year-old HIV-positive man admitted to ICU due to respiratory illness.
To our knowledge no one has previously reported the isolation of A. bereziniae carrying metallo-β-lactamase-resistant blaNDM-1 within the central Sydney region.
Phenotypic identification of non-Acinetobacter species is problematic and challenging, as shown here with A. bereziniae misidentified as A. lwoffi from our Vitek 2 identification results, and also described by other studies with the Vitek MS and MicroScan.
Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS system for the identification of Acinetobacter species from blood cultures: comparison with VITEK 2 and MicroScan systems.
MALDI-TOF MS offers a rapid, convenient identification method for diagnostic laboratories, and can be coupled with other molecular techniques such as 16S rRNA sequencing, rpoB, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridisation, RAPD or whole genome sequencing
Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS system for the identification of Acinetobacter species from blood cultures: comparison with VITEK 2 and MicroScan systems.
Australian Commission on Safety and Quality in Health Care National Alert System for Critical Antimicrobial Resistances (CARAlert): Laboratory Handbook.
in Australia lists Acinetobacter baumannii complex species such as A. calcoaceticus, A. baumannii, A. dijkshoorniae, A. nosocomialis, A. pittii and A. seifertii, as potential organisms carrying carbapenem resistance; however, there is also the worrisome potential role of other non-baumannii Acinetobacter species such as A. bereziniae to carry carbapenem-resistant genes which have implications in infection control in hospital environments. Acinetobacter bereziniae carrying the blaNDM-1 have been shown to carry ISAba elements very similar to multidrug-resistant A. baumannii isolates, with the potential ability to mobilise as a whole and act as reservoirs of blaNDM genes which may contribute to their spreading among clinically relevant Enterobacterales.
We thank Dr Andrew Ginn, the Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Sydney for whole genome sequencing of the isolate and confirming the presence of blaNDM-1.
Conflicts of interest and source of funding
The authors state there are no conflicts of interest to disclose.
References
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Emission of extensively-drug-resistant Acinetobacter baumannii from hospital settings to the natural environment.
Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS system for the identification of Acinetobacter species from blood cultures: comparison with VITEK 2 and MicroScan systems.
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