Project Summary

Genetic Characterization of Salmonella enterica Serovar Newport Isolated from Cattle Using a DNA Microarray to Identify Genes Conveying Multi Drug-Resistance, Increased Virulence and Conjugative Ability

Principle Investigator(s):
Nicole Ramlachan, Toni L. Poole, and David J. Nisbet
U.S. Department of Agriculture, Agricultural Research Service
Completion Date:
May 2006



Non-typhoid Salmonella enterica serovars represent an important health problem worldwide. Estimates of as much as 1.4 million cases of salmonellosis occur each year in the United States (Mead et al., 1999). Salmonella Newport causes significant clinical disease in livestock, particularly cattle, in humans, and in other animal species. Multiple antimicrobial resistant strains of S. Newport have been recorded in the U.S. and Canada. Generally, all of these strains show a degree of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (ACSSuT). In addition, many of these strains show intermediate or full resistance to third-generation cephalosporins, kanamycin, potentiated sulphonamides and gentamicin. Genomic DNA fingerprinting patterns (using pulsed-field gel electorphoresis or PFGE) and antimicrobial resistance profiles have been used to confirm presence of multi drug resistance and group isolates on the basis of phenotype (Fontana et al., 2003; Berge et al., 2004) but this does not provide genotype information or specific on plasmid/integron content. 

S. Newport infections have been increasing at a substantial rate in the US, accounting for 5% of total reported laboratory-confirmed Salmonella infections in 1997 to 10% in 2001 (CDC, unpublished data, 2002). The National Antimicrobial Resistance Monitoring System (NARMS) for Enteric Bacteria has identified an increasing number of S. Newport isolates resistant to at least nine of 17 antimicrobial agents tested: amoxicillin/ clavulanate, ampicillin, cefoxitin, ceftiofur, cephalothin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. These isolates also exhibit decreased susceptibility (minimal inhibitory concentrations (MIC) >16mg/ml) or resistance (MIC >64mg/ml) to ceftriaxone, an antimicrobial agent commonly used to treat serious infections in children. Antibiotic resistance in these strains (referred to as Newport MDR-AmpC) can be attributed to the presence of plasmids containing blaCMY genes which produce AmpC-type enzymes, conferring resistance to penicillin-inhibitor combinations (e.g., amoxicillin/clavulanate), cephamycins (e.g., cefoxitin), and expanded-spectrum cephalosporins (e.g., ceftiofur and ceftriaxone). In 1998, one (1%) of 78 S. Newport isolates tested in NARMS was Newport MDR-AmpC compared with 33 (26%) of 128 in 2001 (CDC, 2002). 

Antimicrobial susceptibility testing does not identify the precise identity of the responsible gene conveying antibiotic resistance. In addition, the use of PCR, though the most frequently used molecular tool to detect antibiotic resistance genes, only allows the detection of a limited number of genes and is not cost-effective on a large scale. In screening bacterial DNA for the presence of multiple antibiotic resistance, virulence and other genes, the DNA microarray technology is a more powerful molecular tool. Previous studies have used microarray detection in the identification of bacterial pathogens, virulence factors, antibiotic resistance genes and genetic polymorphisms (Call et al., 2003; Chen et al., 2005; Chizhikov et al., 2001; Hu et al., 2002; Lee et al., 2002; Volokhov et al., 2002; Westin et al., 2001; Wilson et al., 2002). Protocols for microarray design using published genetic sequence have been standardized and used to screen Salmonella spp. and can be easily used to develop a microarray for specifically screening Salmonella Newport for antibiotic resistance genes, virulence genes as well as additional genes conveying conjugative ability and competitive fitness. 

The stated objectives for this work were: 

To obtain basic knowledge on antimicrobial resistance, virulence and conjugative ability in Salmonella Newport isolated from beef carcasses and hides by genetically determining multi drug resistance status, pathogenicity and ability to transfer and/or uptake such genes in this Salmonella spp. of public health concern.

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