E test

The in vitro susceptibility to antimicrobial agents of the tested E. faecium strain UKI-MB07 and two reference strains E. faecalis ATCC 29212 and E. faecium ATCC 700221 was further tested by determining MIC-values employing the broth microdilution technique according to ISO 20776-1 (2006) as well as Etest (Liofilchem s.r.l., Roseto degli Abruzzi, Italy) according to the EUCAST international guidelines (The European Committee on Antimicrobial Susceptibility Testing [EUCAST], 2014 ). The in vitro susceptibility results of the two reference strains were in the range of published data (European Committee for Antimicrobial Susceptibility Testing [EUCAST] of the European Society of Clinical Microbiology and Infectious Diseases [ESCMID], 2003 ; ISO 20776-1, 2006).

For analysis of the species, bacterial pure culture was subjected to MALDI-TOF (matrix-assisted laser desorption-ionization time of flight) mass spectrometry (MS) (Bruker Diagnostics, Germany) as a duplicate and processed by the routine diagnostic laboratory according to current microbiological diagnostic standards. Species identification was performed as duplicates and a score of >2.0 was considered reliable to the species level [30 (link)]. Antibiotic susceptibility testing was performed using the VITEK^®2 (Biomérieux, Nürtingen, Germany) with the AST-N389 panel and interpreted based on the EUCAST clinical breakpoints (v10.0) for Pseudomonas spp. There are no breakpoints for bacteria of the genus Ralstonia. Since Ralstonia spp. were once assigned to the genus Pseudomonas [31 (link)], clinical breakpoints for Pseudomonas spp. would be the most suitable. MIC (minimal inhibitory concentration) for imipenem and meropenem were confirmed by Etest (Liofilchem, Roseto degli Abruzzi (TE), Italy).

Using the Clinical and Laboratory Standards Institute (CLSI) guidelines [9 ], an antibiogram assay was performed on the isolated K. pneumoniae colonies. The antibiotic discs contained ampicillin (30 µg), cotrimoxazole (25 µg), cefixime (5 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), gentamicin (10 µg), amikacin (30 µg), tetracycline (30 µg), doxycycline (30 µg), minocycline (30 µg), tigecycline (15 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ampicillin/sulbactam (100/10 µg), piperacillin/tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), doripenem (10 µg), aztreonam (30 µg), colistin (10 µg), and fosfomycin (200 µg) (Mast Diagnostics, United Kingdom) [4 , 10 ].
The minimum inhibitory concentration (MIC) of imipenem, meropenem, and colistin for isolates resistant to carbapenems was determined by using E-test (Liofilchem, Italy) according to the 2021 CLSI guidelines [9 ].

To determine the antimicrobial susceptibility patterns of Ochrobactrum sp. POC9, MICs of 10 antimicrobial agents were assessed using Etest™ (Liofilchem, Roseto degli Abruzzi, Italy). The analysis was conducted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [48 ]. The following antibiotics (selected based on bioinformatic analyses that identified putative antibiotic resistance genes) were used: aminoglycosides–gentamicin (CN; concentration of antibiotic: 0.064–1024 µg mL⁻¹), β-lactams (penicillin derivatives)–ampicillin (AMP; 0.016–256 µg∙mL⁻¹), β-lactams (cephalosporins)–cefixime (CFM; 0.016–256 µg∙mL⁻¹), β-lactams (cephalosporins)–cefotaxime (CTX; 0.016–256 µg∙mL⁻¹), β-lactams (cephalosporins)–ceftriaxone (CRO; 0.016–256 µg mL⁻¹), fluroquinolones–ciprofloxacin (CIP; 0.002–32 µg∙mL⁻¹), fluroquinolones–moxifloxacin (MXF; 0.002–32 µg∙mL⁻¹), phenicols–chloramphenicol (C; 0.016–256 µg∙mL⁻¹), ryfamicins–rifampicin (RD; 0.016–256 µg∙mL⁻¹), tetracyclines–tetracycline (TE; 0.016–256 µg∙mL⁻¹). The susceptibility testing was performed at 37 °C for 20 h. After incubation, plates were photographed and MICs were defined. Antimicrobial susceptibility data were interpreted according to the EUCAST breakpoint table (version 8.0) [48 ].

The Etest (Liofilchem, Roseto, Italy) was used to determine the MICs of C. krusei strains in vitro on RPMI-1640 medium following the manufacturer’s instructions (24 (link)). Briefly, the surface of the agar plate was swabbed with the adjusted C. krusei inoculum suspensions (McFarland turbidity of 0.5 at 530 nm). A test strip consisting of a predefined concentration gradient of antifungal agent was placed on the medium surface. After 24 h of incubation, the Etest MICs (μg/mL) were determined directly from the intersection of the growth ellipse and inhibition zone with the tip of the strip. The results of the Etest were verified by M27-A3 microdilution, as proposed by the Clinical and Laboratory Standards Institute (CLSI), with 100 μL diluted suspension (1:50) and 100 μL serial dilutions of each antifungal agent added to each well of 96-well microplates subsequently incubated at 35°C for 24 h.

The antibiotic susceptibility of all the isolates was tested by employing the Kirby-Bauer’s technique as suggested by the CLSI (10 ). The eleven antibiotic disks used include: imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), cefepime (30 μg), cefotaxime (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin/tazobactam (100/10 μg), aztreonam (30 μg) (Mast Group Ltd, UK). Isolates with resistance against a minimum of three groups of antibacterial agents were considered as MDR (11 (link)). To detect ESBL phenotype combined disk method using disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK) was applied to all positively screened isolates by modified hodgE test (MHT) (11 (link)). A growth in the area diameter of ≥5 mm around ceftazidime disc with and without clavulanic acid was expected to be a positive result for ESBL production (12 (link), 13 (link)). The MHT was performed for all isolates as recommended by CLSI (10 ). The E test (imipenem 0.002–32μg/mL) (Liofilchem, Roseto degli Abruzzi, Italy) was applied (according to the manufacturer’s instructions) to all positively screened isolates by PCR test for bla_NDM gene, to determine minimum inhibitory concentrations (MICs).