Tbx agar

Escherichia coli was isolated and identified in accordance with the protocol of the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli from WHO [18 ]. All samples were serially diluted up to 10^-5 dilution in duplicate by using sterile phosphate-buffered saline (PBS; pH 7.4) in a ratio of 1:9. Next, 0.1 mL of the sample from each dilution was added to a petri dish containing tryptone bile X-glucuronide (TBX) agar (Merck, Germany) and plated on the surface using the spread plate method. Bluish-green colonies on the TBX agar plate were suspected to be of E. coli. Petri dishes with a colony count of ≤100 colony-forming unit (CFU)/mL were used for subsequent analysis. Five bluish-green colonies from each TBX agar plate were inoculated into MacConkey agar (MCA; Oxoid, UK) plates. The suspected E. coli colonies on MCA plates appeared as morphologically flat, dry, pink, and nonmucoid colonies with a surrounding darker pink area of precipitated bile salts. The suspected E. coli colony was cultured on a tryptic soy agar medium (Oxoid) and then cultured on sulfide indole motility medium (Oxoid) to perform the indole test to confirm E. coli. The formation of a cherry red ring in the indole test was considered to be a positive confirmation of the E. coli isolate. Escherichia coli ATCC 25922 was used as a positive control.

E. coli was identified in accordance with the International Organization for Standardization guidelines (ISO 16649-2, 2001). Twenty-five grams of the samples were emptied in a stomacher bag (Bag Mixer^®DOA 20550) and added to 225 ml of peptone buffered water. The stomacher bag with the sample was then placed in a bag mixer machine and mashed for 3 min. Afterward, 0.1 ml of the test sample was transferred into the tubes with the use of a sterile pipette. Then, the mixture was incubated at 37°C for 24 h [13 ]. The identification of E. coli was performed in accordance with the International Organization for Standardization guidelines, with the use of the most probable number technique (ISO 16649-2 2003). The tubes exhibiting gas production were recorded as positive, and a loop-full from each positive gas tube was transferred to a separate tube with MacConkey Broth (Oxoid, UK). E. coli confirmation was achieved by observing the gas production and acidification during growth in MacConkey Broth (Oxoid, UK). The positive results were streaked onto tryptone bile glucuronic agar (TBX agar, Oxoid, UK), and the plates were incubated at 37°C for 24 h. The pink colonies were counted using a colony counter-digital machine (Lasec, South Africa) and further subjected to indole and catalase tests.

The presence of E. coli was determined according to ISO 16649-2:2007 [24 ] standard. The same serial dilution was performed as for other determinations (TVC, TYMC, lactic acid bacteria (LAB), and Staphylococcus aureus).
First, 1.0 mL of the diluted sample (10⁻¹ and 10⁻²) was uniformly distributed into a sterile Petri dish; then, TBX Agar (Oxoid, Basingstoke, UK) was poured and mixed. The inoculated dishes were inverted and incubated at 44 °C for 24 h. After incubation, the typical colonies of ß-glucuronidase positive E. coli were counted in each dish (those containing less than 150 typical colonies and less than 300 total colonies) using a colony counter (Colony Star 8500, Funke Gerber, Berlin, Germany). Typical colonies are blue. Non-typical colonies are pale blue. Non-typical colonies found in a Petri dish were also counted and taken into consideration during calculation.

While doing a classical microbiological analysis of the insect samples, E. coli strains were detected and isolated according to standard methods described in DIN EN ISO 16649-2. In brief, samples were subjected to an enrichment step in NaCl-peptone water for 24 h at 37 ± 0.5 °C, and the broth was subsequently streaked on TBX agar (Oxoid, Wesel, Germany) for the selective detection of E. coli.
The species of the isolates was confirmed using a MALDI-TOF biotyper (Bruker Daltonics, Bremen, Germany) and by species-specific PCR targeting the gadA gene [16 (link)]. Only one isolate per sample was included in further investigations.