Incursion of the Dt104 Multiresistance Locus into Salmonella Choleraesuis

The increasing prevalence of multi-drug resistance in pathogenic bacteria is a significant problem for food safety. Salmonella enterica serovar Typhimurium DT104, which is a global health concern and infects a broad range of mammalian hosts, has been shown to carry a chromosomal integron (SGI-1) which encodes multiple antibiotic resistance: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT). The portion of this integron encoding antibiotic resistance is 13 kb and the remaining 30 kb is of unknown function but may contribute to hypervirulence in DT104. The SGI-1 antibiotic resistance gene cluster has subsequently been identified in other serovars suggesting lateral transfer of this element. Host-adapted Salmonella have been assessed for the SGI-1 integron. Isolates of S. choleraesuis, the swine-adapted serovar, with the ACSSuT phenotype have been recovered from pooled clinical isolates. Full sequencing of the SGI1 is underway. Characterization of this integron provides insight into antibiotic resistance, virulence, and SGI-1 transfer between pathogens. Introduction Multidrug resistant (MDR) Salmonella are a major concern in veterinary medicine, food safety, and agricultural practices. Salmonella enterica serovar Typhimurium DT104, which is a global health concern and infects a broad range of mammalian hosts, has been shown to carry a chromosomal integron (SGI-1) which encodes multiple antibiotic resistance: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT). This 43 kb integron is composed of a 13 kb region encoding antibiotic resistance adjacent to 30 kb of unknown function. This larger region contains 15 unknown ORFs as well as genes related to mating pair formation and DNA transfer (Boyd et al., 2001) thus suggesting this is mobile element. In addition, genes in SGI-1 may influence the hypervirulence phenotype observed in DT104 as demonstrated by the correlation between poor clinical outcomes and the presence of SGI-1 (Evans and Davies, 1996; Rasmussen et al., 2005). Our laboratory has recently identified one of the 15 unknown ORFs, hereby designated SGI-1 virulence ORF (SvO), as an activator of hypervirulence for DT104 exposed to protozoa. SGI-1 was originally described in DT104 in 1999 (Briggs and Fratamico, 1999). The SGI-1 antibiotic resistance gene cluster has since been identified in S. enterica serovars Agona (Boyd et al., 2001; Giraud et al., 2000), Paratyphi B (Meunier et al., 2002), Albany (Doublet et al., 2003), Infantis (Carlson et al., 1999), Meleagridis (Ebner et al., 2004), and Newport (Doublet et al., 2004), suggesting lateral transfer of this element. In addition, the entire 43 kb integron was recently conjugally transferred in vitro between S. enterica and an Escherichia coli recipient (Doublet et al., 2005). Given this evidence for horizontal and vertical transfer of SGI-1, its occurrence in additional Salmonella serovars is likely and is of great relevance for therapeutic applications and food safety. While SGI-1 has been found in the aforementioned serovars with broad host ranges, this gene cluster has not been observed in host-adapted serotypes such as Salmonella enterica serotype Choleraesuis. Herein, we assessed S. choleraesuis for the presence of SGI-1. Isolates of S. choleraesuis with the ACSSuT phenotype have been recovered from pooled clinical isolates and DNA sequencing of SGI-1 is underway. Characterization of this integron is essential for assessing implications for antibiotic resistance, virulence, and transmission between pathogens. Materials and Methods Over 300 Salmonella choleraesuis clinical and non-clinical isolates were obtained from the National Veterinary Service Laboratories (Ames, IA). Isolates were pooled and grown in Lennox L broth (Difco) with ampicillin (32 μg/ml, Sigma) and florfenicol (15 μg/ml, Schlering-Plough). The presence of SGI-1 was confirmed using PCR with previously described primers (Carlson et al., 1999). Sequencing of SGI-1 was accomplished using Elongase PCR (Invitrogen) to amplify 6-8 kb regions followed by TOPO pCR-XL cloning (Invitrogen). Primers were designed based on the SGI-1 sequence of S. Typhimurium DT104. PCR conditions were according to manufacturer’s instructions, using the 68° combined annealing and extension. PCR products were visualized using crystal violet and purified by gel excision per manufacturer’s instructions. Colonies were screened for