Eosin methylene blue

Each urine sample was streaked on a freshly prepared differential and selective culture media such as cysteine-lactose-electrolyte-deficient agar, MacConkey agar, mannitol salt agar, eosin methylene blue, and 5% blood agar plates (Oxoid, Ltd, UK) using a calibrated wire loop delivering 0.002 ml of urine. After overnight incubation at 37°C, the plates were inspected for growth and colony characteristics. The incubation was extended up to 48 hours for slow-growing strains. Bacterial colonies differing in size, shape, and color were selected from these plates and separately subcultured in different biochemical tests, which include motility, Gram's reaction, indole tests, methyl red, Voges–Proskauer, citrate utilization, utilization of carbohydrates (such as glucose, sucrose, mannitol, lactose, and fructose), oxidase, catalase, coagulase, and starch hydrolysis test for further characterization and identification [16 ]. A culture was considered significant for UTI if a single bacterium was recovered at a concentration of ≥10⁵ colony-forming unit per milliliter of urine.

A reference strain of EHEC O157:H7 (ATCC 700728) was obtained from Jordan Food and Drug Administration (Amman, Jordan) and was used as a positive control in the study. Bacterial culture was performed according to previously published protocols [3 (link),12 ]. Briefly, fecal swabs were inoculated into 9 mL of Mueller-Hinton broth (Oxoid, UK) and incubated at 37°C for 24 h. Then, a loopful was streaked on MacConkey agar medium (Oxoid) and on eosin methylene blue (Oxoid) and incubated overnight at 37°C. Colonies of E. coli were identified by biochemical reactions using IMViC test (Tulip Diagnostics, India).

Samples were directly streaked onto HiCrome™ Klebsiella selective agar (Himedia, India) at 37 °C for 24 h. The presumptive purple colonies were confirmed by growth on MacConkey’s agar and eosin methylene blue (Oxoid, Cambridge, UK) agar media and identified by biochemical tests [21 ]. The hypermucoviscous phenotype of colonies on an agar plate has been defined by a positive string test [22 (link)]. The 16S-23S rDNA internal transcribed spacer (ITS) of K. pneumoniae isolates were amplified with the species-specific primers reported previously [23 (link)]. K. Pneumoniae isolates grown on Columbia sheep blood agar (Oxoid, Cambridge, UK) plates for 24 h were subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (BioMeri’eux, Marcy I’Etoile, France) identification using αcyano-4-hydroxycinnamic acid matrix solution as previously described [24 (link)]. MALDI-TOF MS running MYLA 3.1.0-15 software (BioM’erieux, Inc., Marcy I’Etoile, France) analyzed, compared the generated spectrum for each tested isolate with a library of standard reference spectra and calculated the confidence values.

From each chicken visceral organ, 2 g was directly inoculated in MacConkey broth and incubated for 18 h at 37°C. Then, a loopful from the previously inoculated broth was streaked onto MacConkey agar (Oxoid) plates for 24 h at 37°C. Rose pink colonies were picked up and streaked onto Eosin Methylene Blue (Oxoid) and incubated overnight at 37°C. The identification of E. coli isolates depends on the colonies morphological characters, and biochemical tests results following Ewing [9 ]. Further identification of E. coli isolates was done using commercial biochemical test kits (bioMerieux API, France).

At enrollment, all children provided whole stool samples, or rectal swabs were used if whole stool collection was not feasible [9 (link)]. These samples were preserved in Cary-Blair media to ensure bacterial viability during transportation for microbiological culture. The samples were then promptly shipped to the central laboratory at the Kenya Medical Research Institute-Centre for Microbiology Research (KEMRI-CMR) in Nairobi within a 24-h timeframe. A swab or a sample of stool was streaked on MacConkey (MAC) (Oxoid, United Kingdom) and Eosin Methylene Blue agars (Oxoid, United Kingdom) and incubated in ambient air at 37 °C for 24 h. Morphologically distinct lactose fermenting mucoid colonies were subcultured onto Mueller Hinton (Oxoid, United Kingdom) agar and subjected to API 20E system (bioMérieux, Inc., France) and oxidase reactions for confirmation of Klebsiella spp. Confirmed Klebsiella spp. isolates were stocked in tryptone soy broth supplemented with 15% glycerol (Oxoid, United Kingdom) and frozen at -80 °C. For this analysis, the Klebsiella spp. isolates were thawed, quadrant streaked for isolation onto MAC agar and incubated at 37° C in ambient air to perform antimicrobial susceptibility testing (AST), DNA extraction and genetic characterization.

Milk samples were diluted at 0.1% peptone water (Oxoid Ltd, England) according to a 1-fold dilution pattern and incubated at 37°C for 24–48 h. Diluted samples were inoculated into McConkey agar (Oxoid, UK) and 5% sheep blood agar using the pour plate method and incubated at 37°C for 24–48 h. All suspected colonies were characterized and picked up for inoculation into buffered peptone water at 37°C for 24–48 h for streaking on specific culture media such as eosin methylene blue (Oxoid) for E. coli, Mannitol salt agar (Oxoid) for Staphylococcus spp., and modified Edwards medium (Oxoid) for Streptococcus spp. Isolates were identified by colony morphology, Gram staining, biochemical characterization, and molecular characterization using polymerase chain reaction with specific genes [20 (link)–22 ].

The study was conducted from February to August 2019. A total of 100 milk samples were collected from different sources (47 samples from dairy farms, 27 samples from retail markets, and 26 samples from farmers’ houses) in Qena, Egypt. These samples were collected under aseptic conditions in a clean, sterile 15 mL falcon tube and transferred instantly in an icebox to the bacteriological laboratory in the Department of Microbiology, Faculty of Veterinary Medicine, South Valley University. One milliliter of milk samples were fed in 9 mL buffer peptone water (Oxoid) and incubated at 37°C for 18-24 h. Subsequently, a sterile loop was used to transfer bacteria from the inoculated buffer peptone water and was inoculated on a MacConkey agar plate (Oxoid). Plates were incubated at 37°C for 24 h. The suspected colonies were inoculated in eosin methylene blue (Oxoid). After the procedure, green metallic sheen colonies were selected for biochemical identification using the IMViC reaction and triple sugar iron test [12 ].