Kolistin direnci olan ESBL'nin araştırılması ve Pseudomonas'ta karbapenem direncinin doğrulanması

Within the scope of the study; MALDI-TOF confirmation of 10 psycrotrophic Pseudomonas whose antibiotic resistance was detected in our previous study (doi.org/10.3390/foods12101956) and the presence of ESBL and colistin resistance in these strains, will be examined.   

Firstly, 10 psychrotrophic Pseudomonas will be transferred to the laboratory under cold conditions. Pseudomonas spp. were transferred to Tryptic Soy Agar (CM 131, Oxoid, Basingstoke, UK) and were incubated at 22 ± 2 °C for 44 ± 4 h. Subsequently, confirmation of carpenem-resistant Pseudomonas strains by MALDI-TOF will be performed in accordance with the EUCAST (2017) standard. MALDI-TOF method has been found in several studies to have good sensitivity and specificity.

EUCAST Guidelines for Detection of Resistance Mechanisms and Specific Resistances of Clinical and/or Epidemiological Importance (2017) will be used to assess. All psychrotrophic Pseudomonas strains will be tested for antibiotic susceptibility by the agar disk diffusion method on Mueller-Hinton agar (CM 337 Oxoid) (EUCAST, 2022). The plates will be incubated at 22±2°C for 24 h. 4 different antibiotics will be used for extended beta-lactamase resistance according to the EUCAST (2017): cefotaxime (CTX; Oxoid, CT0166B, 30 µg), ceftazidime (CAZ; Oxoid, CTO412B, 30 µg), cefoxitin (FOX; Oxoid, CT0119B, 30 µg) and ampicillin (AMP; Oxoid, CTOOO3B, 10 µg). In addition; The double disc synergy test will be used for the phenotypic confirmation of ESBL in Pseudomonas strains (EUCAST, 2017). For this test, paired discs of ceftazidime (30 µg) and cefotaxime (30 µg) will be used, and each was positioned at distances of 20 mm (center to center) from the amoxicillin-clavulanic acid disc (AMC, 20 + 10 µg) (EUCAST, 2017). 

Colistin resistance in 10 antibiotic-resistant Pseudomonas strains will be investigated by disc elution (CLSI, 2021).  For each Pseudomonas strain, 0, 1, 2, and 4 colistin disks of 10 μg will be added into tubes prepared with 10 mm Mueller Hinton broth, respectively. The tubes to which the colistin disk is added will be incubated at room temperature for 30 minutes. Inoculum with 0.5 Mc Farland turbidity will be prepared from Pseudomonas colonies grown on freshly prepared agar. 50 μl of each inoculum will be added to the tubes. The tubes will be vortexed and incubated at 35 °C for 16-20 hours, and then colistin Minimum Inhibitory Concentration (MIC) level will be evaluated (CLSI, 2021).

1.       EUCAST, European Committee on Antimicrobial Susceptibility Testing. Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method. Version 10.0. 2022. Available online: Manual_v_10.0_EUCAST_Disk_Test_2022.pdf (accessed on 26 September 2022).

2.       EUCAST (European Committee on Antimicrobial Susceptibility Testing). EUCAST Guidelines for Detection of Resistance Mechanisms and Specific Resistances of Clinical and/or Epidemiological Importance, Version 2.0. Available online: http://www.eucast.org (accessed on 26 September 2023).

CLSI, Clinical and Laboratory Standards Institute (2021). Performance standards for antimicrobial susceptibility testing, M100, 31st ed. Clinical and Laboratory Standards Institute, Wayne, PA.