Isolation and identification of bacteria

To study antibiotic tolerance of Shigella strain sensitive to ampicillin, we separated a strain sensitive to ampicillin that was originally isolated from Sui County in Henan province in 2014. This strain was confirmed by biochemical and serological identification. Biochemical identification was performed using API20E (API Systems, SA, Vercieu, France). Briefly, the strain was inoculated into Luria-Bertani (LB) agar using a sterile-inoculating loop and incubated at 37°C for 18 hrs. The colonies were picked up with sterile cotton and resuspended in 0.85% NaCl medium to achieve uniform bacterial turbidity, and the turbidity of the bacterial suspension was adjusted to 0.5 McFarland. According to the instructions of the identification manual, the bacterial resuspension was added to the tube of the test strip using a straw. The test strips of citrate utilization(CIT), acetoin production(VP), and gelatinase (GEL) should be filled with bacterial suspension.The test strips of arginine dihydrolase(ADH), lysine decarboxylase (LDC),ornithine decarboxylase (ODC),H2S production(H2S),urease(URE)should be filled with mineral oil to form an anaerobic environment. Sterile distilled water was added to the culture box, and the test strip was placed in a culture box and placed in a 37°C incubator for 18 hrs. The tryptophan deaminase (TDA) test needed to add 1 drop of TDA reagent. The indole production (IND) test needed to add a drop of JAMES reagent. The VP test needed to add a drop of VP1 and VP2. The test results were observed after 10 mins. The test results were interpreted according to the interpretation table, and the results were numerically coded, and the numerical code was found in the identification manual of API20E to determine the type of the strains.

Serological identification was completed using a slide agglutination method. In brief, a clean slide was taken, and a drop of Shigella diagnostic serum was added, and a drop of physiological saline was added as a negative control. Fresh colonies were picked and mixed thoroughly with them. They were allowed to be placed at room temperature for a while, and the flocculation or sand-like agglutination is visible to the naked eye, which is a positive reaction, and vice versa. The results were interpreted according to the test reaction. Strain sensitive to ampicillin was isolated from 39 strains by Kirby–Bauer (K-B) disk diffusion method, as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2017).11 The laboratory number was mel-ss2014011. E. coli ATCC 25922 was used as a control for quality assurance.