Api test system

Twenty-three strains including 15 Salmonella spp and 8 non-Salmonella spp were used for the test. Salmonella isolates included 12 clinical and 3 standard strains belonging to different serovars. Clinical Salmonella isolates were recovered from patients with Salmonella infections admitted to including Children’s Medical Center and Baqiyatallah Hospital in Tehran, Iran, during 2008–2010.
As shown in Table 1, the bacterial positive controls used in this study were S. typhi (ATCC 19430), S. typhimurium (ATCC 14028), S. infantis (clinical strains) and S. enteritidis (ATCC 4931). Additional bacterial pathogens including Campylobacter jejuni (ATCC 33560), Escherichia coli (ATCC 25922), Enterococcus faecalis (PTCC 1393), Klebsiella oxytoca (ATCC 68831), Shigella sonnei (ATCC 9290), Vibrio cholerae (PTCC 1611), Proteus mirabilis (PTCC 1076), and Serratia marcescens (ATCC 14223) were used to check the specificity of the assay.
Identification of the references and clinical strains was confirmed by culture, biochemical testing by the API test system (BioMérieux, Marcy-l’Étoile, France) and slide agglutination with serovar specific antisera (Staten Serum Institute, Copenhagen, Denmark) (5 (link), 28 (link)). All strains were grown on Trypticase Soy Broth (TSB) and incubated at 37 °C for 18 to 24 h to obtain a fresh culture prior to DNA extraction.

Bacillus amyloliquefaciens strain RX7 was identified taxonomically based on phenotypic and physiological characteristics using the Analytical Profile Index (API) test system (Biomérieux, France) and analysis of a partial 16S rDNA sequence. RX7 genomic DNA was prepared from an overnight culture using the phenol-chloroform extraction method. Amplification of the 16S rDNA gene by polymerase chain reaction (PCR) using the F1 (5′-AGAGTTTCCTGGCTCAG-3′) and R3 (5′-AAGGAGGTGATCCAGCC-3′) primers was performed under the following conditions: initial denaturation at 94°C for 5 min, 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 90 s and a final extension at 72°C for 7 min. In addition, a Bacillus subtilis-specific primer set, ytcP-F (5′-GCTTACGGGTTATCCCGC-3′) and ytcP-R (5′-CCGACCCCATTTCAGACATATC-3′), was used to differentiate between B. subtilis and B. amyloliquefaciens under the following conditions: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and primer extension at 72°C for 1 min. Purified PCR fragments were sequenced with both primers and compared with 16S rRNA gene sequences in the public database using BLAST. API CHB50 bacterial identification analysis was also performed according to the manufacturer's instructions.

Three milliliters of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for two minutes at normal speed, using a Stomacher lab blender and incubated at 37°C for 24 hours. A 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for 24 hours. One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli from each sample was streaked on a separate eosin methylene blue agar (EMB agar) plate (Merck, Germmany) and incubated at 37°C for 24 hours. A metallic green colony from each plate with a typical E. coli morphology was selected and examined by biochemical tests, including hydrogen sulfide, citrate, urease and indol based on the API test system (BioMerieux, Marcy L’Etoile, France) (23 ).