Chromid esbl

Sewage Samples. After homogenization, 1 ml of each sample was taken and added to 9 ml of sterile distilled water contained in a screw tube for the preparation of 10⁻¹ dilution. Successive decimal dilutions were made up to 10⁻³and 100 μl of each dilution was then inoculated on McConkey and ChromID^™ ESBL solid media (Biomerieux, France).
Solid Samples. Previously, all food samples were ground in a sterile mortar. 9 ml of sterile distilled water was added to 1 g of each solid sample (sediment/sludge, animal feces, and crushed food item) contained in a screw tube (stock solution). After homogenization through a vortex, 1 ml of the stock solution (10⁻¹ dilution) was added again to 9 ml of distilled water (10⁻² dilution). Successive decimal dilutions were made up to 10⁻⁴. The last three dilutions (10⁻², 10⁻³, and 10⁻⁴) were then seeded on McConkey solid media and ChromID^™ ESBL (Biomerieux, France).
Community Kitchen Samples. Each community kitchen sample swab was cultured on McConkey and ChromID^™ ESBL solid media by streaking with sterile loop (Biomerieux, France).

Samples were inoculated on Brain–Heart Infusion (BHI) broth and incubated at 37 °C for 24 h. All suspensions were then inoculated on a selective chromogenic agar (chromID^® ESBL, bioMérieux, Linda-a-Velha, Portugal) and incubated at 37 °C for 24 h, to isolate the ESBL-positive bacteria. After incubation, the color of colonies was recorded, and bacteria were isolated in BHI agar before further testing.
Isolates were characterized considering their macroscopic morphology on ChromID^®ESBL according to the manufacturer’s instructions (bioMérieux, Linda-a-Velha, Portugal), Gram staining, lactose-fermentation capability on MacConkey agar (Oxoid, Hampshire, UK), and oxidase production. Biochemical identification was performed by IMViC testing [97 (link)] or, in case of inconclusive result by IMViC, API 20E (bioMérieux, Linda-a-Velha, Portugal) galleries were used according to manufacturer’s instructions.

This study implemented three potential one-step AMR testing protocols. These protocols, labeled as Protocol A, Protocol B, and Protocol C, were based on the following agar media: ChromID ESBL (bioMe´rieux), MacConkey agar containing 16 µg/ml of ceftazidime, and MacConkey agar containing 4 µg/ml of cefotaxime, respectively. ChromID ESBL served as an agar designed for isolating and detecting ESBL production. It featured a rich nutrient capacity and an antibiotic, cefpodoxime, known for inhibiting the growth of Gram-positive bacteria and yeast^{53 (link)}. MacConkey agar, on the other hand, was designed to differentiate between fermenting and non-fermenting Gram-negative bacteria. It contained inhibitors for Gram-positive bacteria, crystal violet dye, and bile salts^{54 (link)}. The antibiotics cefpodoxime, cefotaxime, and ceftazidime were recognized indicators of ESBL production (CLSI, version 2021). Antibiotic concentrations followed CLSI guidelines (version 2021). All agars were prepared in-house, except for ChromID ESBL, which was sourced directly from the manufacturer (bioMe´rieux). Subsequently, samples were inoculated onto the three agars and incubated at 37 °C under aerobic conditions for 18 h in accordance with CLSI (version, 2021) recommendations. This approach enabled the assessment of the three protocols’ capability to detect ESBL production within clinical samples.