Study design, study area and period

A community-based cross-sectional study was conducted from January 1st, 2023, to February 30th, 2023, in regular street food vending areas of Arba Minch town, namely: Sikela (nearby bus station), Shecha (nearby Gebeya Dar), and Konso Sefer (nearby Arba Minch General Hospital). Various ready-to-eat street foods are offered on the streets of the town. Arba Minch is the administrative center for the Gamo Zone, which is located in southern Ethiopia, 454 km from Addis Ababa. The city of Arba Minch and its surroundings provide a variety of unique and memorable tourist attractions, and unlike foods in hotels and resorts, the majority of visitors choose street-vended fast food.

Sample collection, culture and identification

A total of 100 ready-to-eat street-vended food samples (100 g from each food item) from four different types of frequently vended and highly consumed street foods, such as “Sambusa” (n = 25), “Ambasha” (n = 25), “Potato Chips” (n = 25), and “Koker” (n = 25), were collected by well-trained sample collectors aseptically using sterile aluminium foil and transported using an icebox within 3–5 °C. Within one hour of being collected, the samples were sliced into smaller pieces with a sterile surgical blade and weighed at 25 g on an electronic digital scale. The measured 25 g samples were homogenised in 225 mL of buffered peptone water (BPW) [Oxoid, Hampshire, UK]. 0.1 mL aliquots from the homogenised or mixed tube were distributed onto MacConkey Agar plates [HiMedia Laboratory Pvt. Ltd., Mumbai, India] and Xylose Lysine Deoxycholate Agar (XLD) [HiMedia Laboratory Pvt. Ltd., Mumbai, India] with an L-shaped sterile wire spreader and incubated at 35 °C for 24 h [6, 14, 15].

Enterobacteriaceae were identified as pink to reddish-purple colonies on MacConkey Agar plates with or without precipitate haloes, while a black colony surrounded by a red colour on XLD was considered to be Salmonella spp. Cell morphology, Gram staining, and various biochemical tests [HiMedia Laboratory Pvt. Ltd., Mumbai, India] were used to characterise the isolated pure colonies of Enterobacteriaceae to the genus and species level, including Triple Sugar Iron Agar (TSI), Simmon Citrate, Indole, Motility, Methyl Red-Voges Proskauer (MR-VP), and Urease, Oxidase, Gas, and H2S production. The pure isolate was subcultured onto nutrient agar plates for confirmatory assays of antibiotic susceptibility, ESβL, and carbapenemase production [15,16,17].

Screening of ESβL and Carbapenemase-producing Enterobacteriacae

ESβL-producing Enterobacteriaceae were first screened for ESβL-production by indicator cephalosporins (cefotaxime (30 μg) and ceftazidime (30 μg)). Isolates that showed an inhibition zone size of ≤27 mm with cefotaxime (30 μg) ≤ 22 mm and/or with ceftazidime (30 μg) were considered potential ESβL-producers and were selected for confirmation for ESβL-production using the Double-Disc Synergy Test (DDST), whereas isolates with reduced susceptibility to meropenem (diameter of the zone of inhibition ≤13 mm), as demonstrated by the disc diffusion method according to the CLSI 2020 guidelines, were considered carbapenem-resistant strains and were selected for confirmation with the Modified Carbapenem Inactivation Method (mCIM) [13, 18,19,20].

Phenotypic confirmation of ESβL by double-disc synergy test (DDST)

Antibiotic discs containing cephalosporins (cefotaxime (30 μg) and ceftazidime (30 μg)) were applied to MHA plate inoculated with a bacterial suspension of 0.5 McFarland standard next to a disc with amoxicillin-clavulanic acid and incubated overnight (18–24 h) at 37 °C. The distance between the discs, 20 mm from centre-to-centre, is optimal for cephalosporin 30 μg discs. The distance between the discs however will be reduced (15 mm) or expanded (30 mm) for strains with very high or low levels of resistance, respectively. A positive result was indicated when the inhibition zones around any of the cephalosporin discs were enhanced or there was a “keyhole” or ≥ 5 mm increase in zone diameter in the direction of the disc of amoxicillin-clavulanic acid [13, 18,19,20].

Modified Carbapenem inactivation method (mCIM)

A loopful of test isolates of the respective species of Enterobacteriaceae (1 μl) was suspended in 2 mL of tryptic soy broth [Oxoid Ltd., Basingstoke, United Kingdom], and a meropenem disc was immersed in the suspension and incubated for a minimum of 4 h at 37 °C. A 0.5 McFarland suspension of E. coli ATCC 25922 indicator strains was prepared in normal saline using the direct colony suspension method and inoculated on an MHA plate using the routine disc diffusion procedure. Then the meropenem disc was removed from the TSB and placed on an MHA plate inoculated with E. coli ATCC 25922 and incubated at 37 °C for 24 h. If the isolate produces carbapenemase, the meropenem in the disc is hydrolyzed, and there is either no zone of inhibition or only limited enhancement in the inhibition zone corresponding to the meropenem-susceptible E. coli (ATCC 25922). Thus, results were judged to be positive for inhibition zones with a diameter of ≤15 mm or presence of pinpoint colonies within a 16–18 mm, and negative for ≥19 mm [18,19,20].

Antibiotic susceptibility testing

Antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method [21], and the results or the diameter of the zone of inhibition were expressed as susceptible, intermediate, or resistant according to the CLSI 2020 guideline after overnight incubation at 37 °C. After preparation of 0.5 McFarland turbidity inoculums, Muller-Hinton Agar (MHA) [Oxoid LTD, Basingstoke, Hampshire, and United England] plates were inoculated, and antimicrobial discs were applied to the plate. The antibiotic discs [Abtek Biologicals Ltd., Liverpool, UK] used in this study were ampicillin (10 μg), amoxicillin-clavulanic acid (20/10 μg), cefotaxime (30 μg), meropenem (10 μg), gentamicin (10 μg), azithromycin (15 μg), tetracycline (30 μg), ciprofloxacin (5 μg), sulfamethoxazole-trimethoprim (3.75/1.25 μg), and chloramphenicol (30 μg). An Enterobacteriaceae isolate was considered multidrug-resistant (MDR) if it was non-susceptible to at least one antimicrobial drug in three or more different classes or groups of antibiotics [19].

Data quality assurance

Reference strains (known for producing ESBL and carbapenemase) obtained from the Ethiopian public health institution (EPHI): E. coli ATCC 25922 and K. pneumoniae ATCC 700603, were used to check the quality of ESBL and carbapenemase test procedures, the quality of culture media, biochemical tests, and the effectiveness of antibiotic discs. Standard operating procedures (SOP) were strictly implemented from sample collection up to final microbiological identification. All culture media were prepared following the manufacturer’s instructions, and the sterility of the culture media was tested by incubating 5% of the batch at 35–37 °C overnight for evaluation of possible contamination.