Xylose lysine deoxycholate agar

Pectin (63–66% degree of esterification, Mw = 30,000–100,000 g/mol), polyethylene glycol 400 (PEG₄₀₀), zinc nitrate, and magnesium nitrate hexahydrate were purchased from Loba Chemie, India. Anhydrous calcium chloride and sodium chloride were provided by Fisher Scientific, UK. Microbiological media, including Tryptic Soy Broth (TSB), Mueller–Hinton Agar (MH), Xylose Lysine Deoxycholate agar (XLD), and Mannitol Salt Agar (MSA), were provided by HiMedia (India). All solvents were of analytical grade and obtained from recognized chemical suppliers.

Stool specimens received at respective laboratories were cultured for enteric pathogens, including Shigella spp. by standard methods [17 ]. Sample was streaked onto MacConkey agar and a selective medium (either deoxycholate citrate agar or xylose lysine deoxycholate agar or Salmonella-Shigella agar) (all media by HiMedia, India) by the quadrant isolation technique [18 ]. The plates were incubated at 37 °C for 24 h. Isolates were identified by Gram’s staining, colony characteristics, and biochemical tests.
After identification of isolates, they were subcultured onto nutrient agar slants in screw capped tube, incubated overnight and then transported to NPHL in a cold box. All reported isolates of Shigella were re-confirmed by serotyping using commercially available antisera from Denka Seiken, Japan, and were preserved in tryptic soy broth with 20 % glycerol at −75 °C for further use. Antibiotic susceptibility test was performed for all the identified Shigella isolates by Standard Kirby Bauer’s disc diffusion technique. The antibiotics used for analysis were ampicillin (Amp-10 mcg), cotrimoxazole (Sxt-25 mcg), nalidixic acid (NA-30 mcg), ciprofloxacin (Cip-5 mcg), mecillinam (Mec), and ceftriaxone (CRO-5 mcg).

Salmonella isolation was conducted according to Global Foodborne Infections Network, laboratory protocol [17 (link)]. Briefly, 1 g of stool sample was suspended in 9 ml of buffered peptone water (Himedia, India) and incubated for 24 hours at 37°C. Then, 100 μl of the suspension was transferred to 10 ml of Rappaport Vassiliadis enrichment broth (RVB), (Oxoid, UK) and incubated for 24 h at 42°C. Suspension of 1 ml of each sample was also transferred to 9 ml of Muller-Kauffmann-Tetrathionate broth (Himedia, India) and Selanite-F broth (Becton-Dickinson, USA) and incubated for 24 h at 37°C. Samples from these three enrichment broths were streaked on to Xylose-Lysine Deoxycholate Agar (Himedia, India), and the plates were incubated at 37°C for 24-48 hours.
Biochemical tests were conducted for presumptive Salmonella colonies using Urea, Triple Sugar Iron Agar, Lysine Iron Agar, and Citrate as described elsewhere. Typical Salmonella colonies were confirmed using genus specific PCR as described previously [18 (link)]. Serotyping of Salmonella isolates was conducted at the National Microbiology Laboratory, Office International des Epizooties (OIE) Salmonella Reference Laboratory, Public Health Agency of Canada. Somatic (O) antigens were determined using slide agglutination tests whereas flagellar antigens using microplate agglutination technique [19 , 20 (link)].