*2.4. Detection of Integrons and Antibiotic Resistance Genes*

The isolates that were classified as resistant according to the results of the disc diffusion test (*n* = 54) were screened by PCR for the most relevant AMR genes corresponding to their phenotypic resistance pattern. In addition, all resistant isolates were screened by PCR for the presence of integron class 1 and 2 encoding genes. The isolates tested were *S.* Newport (*n* = 18), *S.* Bolton (*n* = 8), *S.* Hadar (*n* = 6), *S.* Mbandaka (*n* = 4), *S.* Heidelberg (*n* = 8), *S.* Typhimurium (*n* = 2), and *S.* Zanzibar (*n* = 8) serotypes. The existence of class 1 integron was investigated by PCR for the detection of genes encoding the variable part between the 5' conserved segment and the 3' conserved segment of the variable region [33]. Presence of class 2 integron was investigated by detection of *hep74* and *hep51* genes using primers and following PCR conditions previously reported [33]. Presence of 22 AMR genes (Table 1) known to confer resistance to six commonly used classes of antimicrobials (β-lactams, tetracyclines, phenicols, fluoroquinolones, trimethoprim, and sulfonamides) were investigated by PCR. The primer sets used for detection of integrons and AMR genes are shown in Table 1. Ampicillin resistant isolates (*n* = 4) were screened for four β-lactamase resistance encoding genes, and ciprofloxacin resistant isolates (*n* = 40) were screened for four fluoroquinolone plasmid mediated quinolone resistance (PMQR) determinant genes. Chloramphenicol resistant isolates (n=4) were screened for four phenicol resistance genes, tetracycline resistant isolates (n=12) were screened for three genes. Sulfonamide resistant isolates (*n* = 21) were screened for two genes and six trimethoprim resistant isolates were screened for five trimethoprim resistance genes. These genes were selected because they are the most frequently detected genes associated with the corresponding phenotypes of the NTS isolates [34]. All the integron PCR products were purified and sequenced (GATC Biotech, Cologne, Germany) and the sequence results were analysed using BLAST and compared to GenBank database (http: //blast.ncbi.nlm.nih.gov/blast.cgi). Similarly, one PCR product from each of the AMR PCRs was sequenced to confirm the PCR results. Negative controls were included in all PCR analyses.


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The β-lactamase encoding genes (*blaPSE-1, blaCMY-2, bla*TEM-1, *blaOxA*) encode production of β-lactamase enzyme that breaks the β-lactam antibiotic ring open and deactivates the molecule's antibacterial properties. The plasmid mediated quinolone resistance genes (*qnrA, qnrB, qnrC, qnrS*) encode pentapeptide repeat proteins that bind to and protects DNA gyrase and topoisomerases IV from the inhibition of quinolones. The phenicol resistance genes, (*cat1, cat2*) encode chloramphenicol acetyltransferase enzyme that inactivates chloramphenicol, chloramphenicol resistance gene, *cmlA* and florfenicol resistance gene *floR*, encode efflux pump proteins. Sulfonamide resistance genes *sul1* and *sul2* encode insensitive sulfonamide-resistant dihydropteroate synthase which cannot be inhibited by sulfonamide. Tetracycline resistance genes (*tetA, tetB, tetG)* encode membrane associated efflux pump proteins that export tetracycline from the cell and reduces drug concentration and thereby protecting ribosomes. Trimethoprim resistance genes *(dhfrI, dhfrV, dhfrVII, dhfrIX, dhfrXIII*) encode a drug-insensitive dihydrofolate reductase which cannot be inhibited by trimethoprim.
