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Antimicrobial susceptibility testing

The antimicrobial susceptibility and MICs were determined by broth microdilution method using the MIKRO-LA-TEST MIC NEFERM kit according to the manufacturer's instructions (Erba Lachema, Brno, Czech Republic) in triplicates. The results were evaluated according to the CLSI.1, 2 In order to evaluate multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) phenotypes,3 susceptibility to tetracycline was tested too, using Etest (bioMérieux). The susceptibility to tetracycline was tested only in isolates carrying the tet(B) gene, for which resistance to tetracycline would alter the phenotype from XDR to PDR. Two isolates (AB14-VUB and AB189-VUB) not carrying genes conferring resistance to tetracycline were included as a negative control.

Please cite:
Valcek A, Nesporova K, Whiteway C, De Pooter T, De Coster W, Strazisar M, Van der Henst C. Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii. Antimicrob Agents Chemother. 2022 Aug 15:e0089222. doi: 10.1128/aac.00892-22. Epub ahead of print. PMID: 35969073.

Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was used for direct capsule visualization by labelling the capsule of 11 A. baumannii isolates, 9 modern clinical isolates and 3 established strains, which ranges from high to low densities. The fixation and staining of the bacteria were performed as described before.4 The cupule with the fixed pellet of bacteria (polymerized for 5 hours) was embedded in resin and polymerized (12h at 37°C, 48h at 45°C and 3 days at 60°C). The ~60 nm slides of the resin were marked with acetate uranyl and placed on the electron microscopy grid.

Please cite:
https://www.biorxiv.org/content/10.1101/2022.02.27.482139v3

Macrocolony type morphology

5 µl of overnight bacterial suspension (~ 1x108 cells) was plated on Columbia Agar with 5% Sheep Blood purchased from BD (Becton, Dickinson and Company, Franklin Lakes, NJ). The plates were incubated non-inverted for 6 days at 25°C and subsequently photographed by a Canon® camera.

Please cite:
https://www.biorxiv.org/content/10.1101/2022.02.27.482139v3

Galleria mellonella model of infection

TruLarv research grade larvae of G. mellonella (BioSystems Technology) were stored at 15°C no longer than 5 days after arrival and were incubated for 30 min at 4°C prior to injection. Bacteria from an overnight culture were washed with physiological saline (PS) (0.9% NaCl) and diluted to approx. 1x107 CFU/ml. The larvae were injected with 10 µl of PS containing 1x105 CFU/ml of A. baumannii in the last left proleg using a 0.3 ml insulin syringe (BD MicroFine). Each of the nine selected strains of A. baumannii were injected into 10 larvae and 10 larvae were injected with PS as a negative control. The experiments were carried in duplicates and the survival (assessed by keratinization and mobility) rate was evaluated each day in a period of 5 days.

Please cite:
https://www.biorxiv.org/content/10.1101/2022.02.27.482139v3

Density gradient

1 ml of overnight culture in a 1.5 ml microtube was centrifuged for 2 min at 7000 rcf. The supernatant was removed, and the pellet was resuspended in 1 ml of PBS. Subsequently, 875 µl of PBS resuspended bacteria were mixed with 125 µl of LUDOX® LS colloidal silica (30 wt. % suspension in H2O, Merk).5, 6 This mix was then centrifuged for 30 min at 12.000 rcf and immediately photographically recorded.

Please cite:
https://www.biorxiv.org/content/10.1101/2022.02.27.482139v3

References

  1. CLSI. 2018. Performance standards for antimicrobial susceptibility testing. CLSI document M100. Clinical and Laboratory Standards Institute, Wayne, PA.
  2. CLSI. 2018. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI document M07. Clinical and Laboratory Standards Institute, Wayne, PA.
  3. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. 2012. Multidrug- resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281. https://doi.org/10.1111/j.1469-0691.2011.03570.x.
  4. Hernando-Amado S, Coque TM, Baquero F, Martínez JL. Defining and combating antibiotic resistance from One Health and Global Health perspectives. Nat Microbiol. 2019 Sep;4(9):1432-1442. doi: 10.1038/s41564-019-0503-9. Epub 2019 Aug 22. PMID: 31439928.
  5. Ardissone S, Fumeaux C, Bergé M, Beaussart A, Théraulaz L, Radhakrishnan SK, Dufrêne YF, Viollier PH. Cell cycle constraints on capsulation and bacteriophage susceptibility. Elife. 2014 Nov 25;3:e03587. doi: 10.7554/eLife.03587. PMID: 25421297; PMCID: PMC4241560.
  6. Kon H, Schwartz D, Temkin E, Carmeli Y, Lellouche J. Rapid identification of capsulated Acinetobacter baumannii using a density-dependent gradient test. BMC Microbiol. 2020 Sep 16;20(1):285. doi: 10.1186/s12866-020-01971-9. PMID: 32938408; PMCID: PMC7493399.