Antimicrobial resistance and class 1 integrons in Salmonella enterica subsp. enterica serovar Derby isolates from pig abattoirs

Salmonella enterica subsp. enterica (S.) serovar Derby is one of the most prevalent serovars in pigs. The aim of this study was the investigation of S. Derby isolates for the presence of antimicrobial resistance and class 1 integrons and their gene cassettes. Forty-nine S. Derby isolates, obtained from different sources at four pig abattoirs (A-D) in Southern Brazil were analysed. Five isolates were susceptible to all antimicrobial agents tested. Twenty-seven isolates were multi-resistant, showed a common resistance pattern to streptomycin/spectinomycin-sulphonamides-tetracycline and shared the same XbaI-pattern. Except for one isolate, all these multi-resistant isolates carried class 1 integrons. The integrons are most likely located in the chromosomal DNA of 16 and ten S. Derby isolates from abattoirs A and B, respectively. All amplicons for the variable part of class 1 integron showed the same EcoRI RFLP pattern. The integrons harboured a new aadA variant designated aadA26, which encodes combined resistance to streptomycin and spectinomycin. The presence of the same class 1 integron among related isolates, from the same or different abattoirs, points towards a dissemination of the integron by a clonal expansion of the isolates. Introduction Salmonella enterica subsp. enterica serovar (S.) Derby has been commonly isolated from slaughter pigs and pork products (Hauser et al., 2011). In 2011, S. Derby was among the top five serovars most frequently isolated from clinical and non-clinical isolates from non-human sources, which were submitted to the National Veterinary Services Laboratories (NVSL) in the U.S.A. Moreover, it was the second most isolated serovar from porcine sources (CDC, 2013). In Southern Brazil, this serovar was also among the most common serovars isolated from pigs and pork products (Bessa et al., 2007; Mürmann et al., 2009). Multi-resistant (resistance to three or more classes of antimicrobial agents) S. Derby isolates have been obtained from different sources, and integrons with different gene cassette arrays have been identified in this serovar (Akiba et al., 2006; Beutlich et al., 2011). Integrons are genetic elements able to integrate and excise gene cassettes by site-specific recombination: They usually carry antimicrobial resistance gene cassettes and therefore contribute to maintenance and dissemination of antimicrobial resistance. The aim of this study was the investigation of S. Derby isolates from pigs for (a) the presence of antimicrobial resistance, (b) the detection of class 1 integrons and their gene cassettes and (c) the location of the class 1 integrons. Material and Methods A total of 49 S. Derby isolates obtained from lairage, pig carcasses and intestinal contents at four abattoirs (A-D) in 2008 in Southern Brazil were analyzed. They were tested for susceptibility to 12 antimicrobial agents by agar disk diffusion (CLSI, 2008). Multi-resistant isolates (n=27) were further investigated by XbaI-macrorestriction analysis (Ribot et al., 2006), plasmid profiling (Schwarz and Liebisch, 1994), and PCR assays for the detection of the resistance genes: sul1, sul2 and sul3 (sulphonamide resistance), tet(A) and tet(B) (tetracycline resistance) and strA (streptomycin resistance) and aadA variants (streptomycin/spectinomycin resistance) (Frech et al., 2003; Kadlec et al., 2005). Integrons were screened by PCR assays for the presence of the intI1 integrase gene and the variable part of class 1 integrons (Sandvang et al., 2007). The amplicons specific for the variable part of class 1 integrons were analysed by restriction fragment length polymorphism (RFLP) using the EcoRI restriction enzyme. A representative amplicon was chosen for cloning into pCR®2.1TOPO Vector (Invitrogen, Groningen, The Netherlands) and the recombinant vector was transformed into Escherichia coli recipient strain TOP10. Sequence analyses were conducted with the M13 forward and reverse primers (MWG, Ebersberg, Germany). Sequence comparisons were carried out using the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/) and with the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The nucleotide sequence of the amplicon for the variable part of class 1 integron has been deposited in the European Molecular Biology Laboratory (EMBL) database under the accession number HG314953.1. To confirm the linkage between aadA26 and sul1, as well as, aadA26 and qacED1, specific PCR assays were used (Michael et al., 2005).