Food Protection Trends - May 2010 - 274

INTRODUCTION
Foodborne diseases caused by nontyphoid Salmonella represent an important public health problem and an economic burden in many parts of the world today (11, 18, 27). In the United States (US), Salmonella is the second most common identifiable cause of illness, and the leading cause of hospitalizations and deaths, due to foodborne bacterial infection (17). Most people who suffer from Salmonella infections present with temporary gastroenteritis that usually does not require treatment. However, when infection becomes invasive, antimicrobial treatment is mandatory (29). Traditionally, ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole have been used to treat such severe cases. However, the increasing number of antimicrobial-resistant Salmonella strains has led to a decrease in the efficacy of these treatments (2). Additionally, the frequency of isolation of Salmonella strains resistant to one or more antimicrobial agents has risen in the US (7), and elsewhere in the world (1). Fluoroquinolones and broad-spectrum cephalosporins have been employed most recently as the preferred drugs for treatment of adults and children, respectively, due to the low likelihood of resistance to them (2, 4). However, the usefulness of these drugs may be diminishing, as Salmonella strains producing β-lactamases conferring resistance to broad-spectrum cephalosporins have been isolated from clinical patients (6, 29); some of these microorganisms have been acquired from cattle (8). The situation is reported to be more complex and difficult in developing countries in which there is a widespread misuse of antimicrobials in both human and veterinary medicine practices (21). This uncontrolled exposure to combinations or several classes of antimicrobials has led to the emergence of multidrug-resistant (MDR) strains that may pass from food animals to humans (7). The spread of antibiotic resistance among bacteria has been associated with mobile genetic elements such as plasmids, transposons (30) and integrons (19). Notably, MDR has been frequently linked with microbial genomic elements known as integrons, which have the ability to distribute genes encoding resistance to a number of antimicrobial drugs (19). Integrons can capture genes, notably those encoding antimicrobial resistance,

by a site-specific recombination system and have been located in both chromosomal and extra chromosomal DNA (3, 12). The main classes of integrons are found in the family Enterobacteriaceae, with class 1 integrons being the most extensively studied. Class 1 integrons are characterized by the presence of two conserved segments, the 5'-conserved segment (5'-CS) and 3'-conserved segment (3'-CS) (3), and are defined by an intI gene encoding integrase, a recombinant site attI, and a strong promoter. Previous studies (30, 31) on integrons and associated antimicrobial resistance genes in Salmonella revealed a predominance of gene cassettes that confer resistance to aminoglycosides and trimethoprim. The investigation of multidrug-resistance in foodborne pathogens in general and Salmonella in particular is essential for a more complete understanding of the epidemiology of emerging multidrug resistance in Salmonella serovars (31). The implication of therapeutic failure in public health due to multidrug resistance is particularly important, given that Salmonella is the leading cause of foodborne infection in the US (17). This study reports on the association between the presence of integrons (Class 1 and 2) and MDR in Salmonella serovars isolated from naturally infected feedlot cattle housed at the North Dakota State University (NDSU) cattle feedlot research facility.

inson & Company MD, USA) supplemented with cefixime and potassium tellurite (Dynal Biotech ASA, Oslo, Norway). Both the fecal samples and swabs were placed in iced-pack coolers before being transported to the laboratory. The sampling procedure was repeated every three weeks for the entire finishing period (March – June, 2007) and has been described in detail elsewhere (26).

Isolation of Salmonella
Fecal samples were cultured by use of conventional culture methods optimized for the detection of Salmonella (14). Briefly, a sterile swab was loaded with fecal sample, which was pre-enriched in buffered peptone water (Difco™ Becton Dickinson & Company, MD) at 37°C overnight; this was followed by immunomagnetic bead separation specific for Salmonella species (Dynabeads® anti-Salmonella, Dynal Biotech, Inc., Lake Success, NY) according to the manufacturer’s instructions. After the final wash, the beads were transferred to 10 ml of Rappaport Vassiliadis R10 (RV) broth (Becton Dickinson, Sparks, MD) and incubated (with constant gentle shaking) at 42º for 24 h. Following incubation, the RV cultures were streaked onto modified brilliant green agar (mBGA) (Becton Dickinson) and mannitol lysine crystal violet brilliant green agar (MLCB) (Oxoid LTD, Basingstoke, UK). Colonies with typical Salmonella characteristics were stabbed and also inoculated on the surface of 10 ml triple sugar iron agar slants (Becton Dickinson), and biochemical results were read after 24 hours incubation.

MATERIAL AND METHODS
Sample collection
Fecal samples were collected from each steer in accordance with the guidelines established by the Institute for Animal Care and Use Committee (IACUC), following a previously described protocol (13). Briefly, each steer was restrained in a hydraulic chute, and about 20 g of feces was collected from the rectum. A new set of sterile polythene sleeve gloves was used for collection from each steer. The feces were put into sterile plastic cups that were placed on ice to be transported to the laboratory at NDSU for processing. In addition, a sterile dry cotton swab was used to culture the dorsal mucosa of the rectoanal junction (RAJ). Each RAJ mucosal swab was placed into a 15 ml culture tube containing 3 ml of Trypticase Soy Broth (Difco™ Becton Dick-

Serotyping and antimicrobial susceptibility testing
Serotyping of Salmonella isolates was performed at the National Veterinary Laboratory Services (NVSL) at Ames, Iowa, following standard methods. The antimicrobial susceptibility test of Salmonella isolates was determined on a panel of 15 selected antimicrobials by use of the Sensititre automated antimicrobial susceptibility test system (TREK Diagnostic Systems, Westlake, OH) according to the manufacturer’s instructions. The MIC breakpoint levels and concentrations of each antimicrobial were based on those specified by the

274 FOOD PROTECTION TRENDS | MAY 2010

Food Protection Trends - May 2010

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