Acinetobacter

Acinetobacter genome assemblies from our collection for which the KL and OCL types had been previously determined via manual or automated sequence inspection ([2, 41 (link)]; and unpublished data) were used to assess the level of typing accuracy that could be achieved through the use of our novel databases with Kaptive. Paired-end Illumina read data (described in [2, 41 (link)] and available under BioProject accession PRJEB2801) were de novo assembled using SPAdes v. 3.13.1 [46 (link)] and optimized with Unicycler v. 0.4.7 [47 (link)]. High-quality genome assemblies (n=719) with a maximum contig number of 300 and minimum assembly length of 3.6 Mbp were included in the analysis (cut-offs determined empirically by manual inspection of the contig number and assembly length distributions, respectively). These assemblies were assessed for oxaAb presence using blastn (>95 % nucleotide sequence similarity and >90 % combined coverage) to confirm the A. baumannii species assignment. Confirmed A. baumannii sequences (n=642) were analysed using both KL and OCL reference databases with command-line Kaptive v. 0.7 [44 (link)] with default parameters.
The same method was used to test databases against 3412 genome sequences available in the NCBI non-redundant and WGS databases as of February 2019. These genome assemblies were bulk downloaded from NCBI as a compressed .tar file for local analysis. Genomes lacking oxaAb were removed prior to typing but quality control (QC) analysis as described above was applied to this data set only after typing was complete.

For validation of the CIM, a selection of 30 Gram-negative isolates was used. This selection included isolates obtained from different institutes across the world carrying known carbapenemase encoding genes and carbapenem susceptible isolates, according to the submitter (Table 1). In addition, 694 isolates submitted to the National Institute for Public Health and the Environment for the national surveillance of carbapenemase-producing Enterobacteriaceae (CPE) by Dutch medical microbiology laboratories (MMLs) during the first six months of 2012 and the first six months of 2013 were used. For the national surveillance of CPE in the Netherlands, Dutch MMLs are requested to submit Enterobacteriaceae isolates with an MIC for meropenem > 0.25 μg/ml. However, more than half of the isolates (411/694, 59%) sent in for CPE surveillance were non-fermenting Gram-negatives belonging to the genera Pseudomonas and Acinetobacter. Furthermore, 35% of the isolates had MICs below 0.25 μg/ml. Nevertheless, all isolates were included in this study.
The species identification, as performed by the MMLs, was confirmed using MALDI-TOF (Bruker Daltonics GmbH, Bremen, Germany) and the MIC for all isolates was confirmed by E-test (BioMerieux Inc., Marcy L’Etoile, France). Culturing of isolates was done on Columbia Sheep Blood (bioTRADING Benelux BV, Mijdrecht, The Netherlands) and Mueller-Hinton agarplates (Oxoid Ltd, Hampshire, United Kingdom). An overview of all CPE surveillance isolates and their characteristics is displayed in Tables 2 and 3.

Clinical samples from various illnesses in the ICU (n = 79) were prepared for culture using traditional techniques (cultures on routine media such as blood agar or MacConkey agar and selective media with specific biochemical tests when necessary) where all the clinical isolates have proceeded for culture on blood and MacConkey agars and then Gram staining was performed to identify bacterial morphology and Gram reaction. The studies were cultured several times to ensure that all isolates were pure. Several biochemical tests were performed to confirm that all isolates belonged to A. baumannii, including oxidase, catalase, and indole tests. Standard phenotypic assays were used for the initial identification [25 (link)].
Vitek 2 system (BioMerieux, Marcy-l’Étoile, France) was used in the microbiology laboratory to confirm the identification of the isolates, following the manufacturer’s instructions. All isolates were investigated for antibiotic susceptibility, using this automated Vitek 2 Compact system. The included samples were cultured on blood agars, and then the suspension was made for every single isolate. A liquid suspension of the studied isolates was loaded on the Vitek system, and left overnight to obtain the result. The next day, results illustrated the samples’ identification and antibiotic susceptibility.
VITEK 2 system was approved for authenticating the names of Acinetobacter spp. as described by the manufacturer (BioMerieux). The Vitek2 card contains 64 wells holding different fluorescent biochemical assays. Twenty out of the sixty-four wells were carbohydrate assimilation; four are phosphatase, nitrate, urea, and actidione tests. The machine controlled this card automatically, including filling, sealing, and finally transferring such cards into the linked incubator at a temperature of 35 °C. Each output report is usually decoded according to a specific algorithmic system. The acquired results were recognized and recorded. Most known Acinetobacter spp. have clear-cut profiles, and the system led to the correction of the unknown organism.
The susceptibility of the most commonly used antibiotics (n = 13) for the prevalent ICU infections has been recorded.

Susceptibility testing was performed by the Kirby–Bauer disk diffusion method. Overnight cultures were suspended in physiological saline to 0.5 McFarland units (McFarland Densitometer DEN-1, Biosan, Latvia). The suspension was inoculated on Mueller–Hinton agar (Oxid, UK). Selected antibiotics were placed on the inoculated plates. For S. aureus strains, cefoxitin 30 µg, ceftriaxone 30 µg, benzylpenicillin 1iu, ampicillin 2 µg, ampicillin–sulbactam 10/10 µg, amoxicillin–clavulanic acid 20/10 µg, norfloxacin 10 µg, amikacin 30 µg, erythromycin 15 µg, clindamycin 2 µg, and chloramphenicol 30 µg were applied (Liofilchem, Italy). For K. pneumoniae and Serratia liquefaciens strains, amoxicillin–clavulanic acid 20/10 µg, piperacillin–tazobactam 30/6 µg, cefotaxime 5 µg, ceftazidime 10 µg, ertapenem 10 µg, imipenem 10 µg, meropenem 10 µg, ciprofloxacin 5 µg, gentamicin 10 µg, and trimethoprim/sulfamethoxazole 1.25/23.75 µg were applied (Liofilchem, Italy). For Acinetobacter spp., piperacillin–tazobactam 30/6 µg, ceftazidime 10 µg, imipenem 10 µg, meropenem 10 µg, ciprofloxacin 5 µg, and amikacin 30 µg were applied (Liofilchem, Italy). For Acinetobacter spp., imipenem 10 µg, amikacin 30 µg, gentamicin 10 µg, trimethoprim/sulfamethoxazole 1.25/23.75 µg, ciprofloxacin 5 µg, and levofloxacin 5 µg were applied (Liofilchem, Italy). The size of the zone of inhibition around the disk was measured after 16–20 h of incubation. The evaluation of the results was carried out according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard, actual EUCAST version [17 ].