Sensititre system

Kirby-Bauer (KB) disk diffusion assay was conducted to determine antibiotic resistance profiles using antibiotic paper disks (BD Technologies). The antibiotics used in KB test were amikacin (30 μg), amoxicillin with clavulanic acid (20/10 μg), chloramphenicol (30 μg), kanamycin (30 μg), nalidixic acid (30 μg), sulfamethoxazole with trimethoprim (23.75/1.25 μg), sulfisoxazole (0.25 μg), and tetracycline (30 μg). Susceptibility or resistance profiles were interpreted according to Clinical Laboratory Standards Institute (CLSI) guidelines. Extended spectrum β-lactamase (ESBL) sensitivity profiles were screened using confirmatory ESBL plates according to manufacturer’s instructions (Sensititre; Thermo Fisher Scientific, Oakwood Village, OH, USA). The dilution range of antibiotic concentrations used were as follows; cefazolin (8–16 μg/ml), cefepime (1–16 μg/ml), cefoxitin (4–64 μg/ml), meropenem (1–8 μg/ml), cephalothin (8–16 μg/ml), cefpodoxime (0.5–64 μg/ml), ceftriaxone (1–128 μg/ml), ciprofloxacin (1–2 μg/ml), gentamicin (4–16 μg/ml), ampicillin (8–16 μg/ml), imipenem (0.5–16 μg/ml), piperacillin/tazobactam (4/4–64/4 μg/ml), ceftazidime (0.25–128 μg/ml), ceftazidime/clavulanic Acid (0.25/4–128/4 l μg/ml) and cefotaxime (0.25–64 μg/ml). Plates were incubated at 37 °C for 18 h and read automatically using Sensititre System (Thermo Fisher Scientific).

The identification of etiology was based in accordance with local laboratory techniques. From positive cultures, Gram staining and a rapid identification protocol were adopted. The bacterial pellet obtained directly from positive cultures was used for MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA) identification and for molecular analysis. The SensiTitre™ system (Thermo Fisher Scientific, Waltham, MA, USA) or the Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France) were used for isolate identification and antimicrobial susceptibility testing. Minimum inhibitory concentrations (MICs) were established according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [12 ,13 ]. A multiplex PCR, combined with array detection, using an automated closed system that isolates, amplifies, and detects nucleic acid for multiple causative pathogens within a single specimen in one step (FilmArray, BioFire Diagnostics, BioMérieux, Salt Lake City, UT, USA) was used, from 2018, to identify the possible organisms and the corresponding bacterial resistance genes within 60–90 min.

The EUCAST standard DD method was performed in parallel (i.e., from subcultured colonies and zone readings after 16 to 20 h of incubation) with media and disks from Oxoid/Thermo Fisher Scientific. The results from the standard DD method were used as the reference (23 ). Categorization was performed according to the EUCAST breakpoint table for standard DD testing valid at the time of the study (versions 10.0 and 11.0) (24 ). The EUCAST DD results were recorded by the laboratory staff in the LIS. Selected isolates were further analyzed by broth microdilution (BMD) using the Sensititre system (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.

From positive blood cultures, Gram staining and a rapid identification protocol were adopted [27 (link)]. The bacterial pellet obtained directly from positive blood cultures was used for MALDI-TOF MS (Bruker Daltonics) identification and for molecular analysis. The presence of a bla_KPC gene was determined by PCR using the GeneXpert® System (Cepheid). Antimicrobial susceptibility tests were performed with the SensiTitre™ system (Thermo Fisher Scientific) or Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Minimum inhibitory concentrations (MICs) were classified according to breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [28 ].

For the implementation of the MIC testing, the strains to be tested were plated out on Columbia blood agar (Biomérieux) and incubated under microaerophilic conditions 44 ± 4 h at 37 ± 1°C. This culture was used to prepare a suspension according to McFarland 0.5. From this suspension, 50 μL was transferred in Mueller‐Hinton broth with TES/lysed Horse Blood (TREK Diagnostic Systems; Thermo Scientific, Waltham, MA, USA). The resistance determination was performed using the Sensititre^® system (TREK Diagnostic Systems; Thermo Scientific), a technique for determining the MIC value. For this purpose, dehydrated AB gradients were applied to the wells of a microtiter plate, with the appropriately prepared bacterial suspension inoculated (with automatically inoculator) and incubated 44 ± 4 h at 37 ± 1°C under microaerophilic conditions. For the evaluation of the microtiter plates, SensiTouch^® system was used. In this system, step by step, each AB gradient was accessed. The following antibiotics were used: ampicillin, amoxycillin/clavulanic acid, chloramphenicol, ciprofloxacin, colistin, erythromycin, gentamycin, imipenem, nalidixic acid, neomycin, streptomycin, tetracycline.